Optogenetic probes for measuring membrane potential

ABSTRACT

The invention provides methods, cells and constructs for optical measurement of membrane potential. These methods can be used in cells that are not accessible to presently available methods using electrodes. The methods can be directed to, for example, high-throughput drug screening assays to determine agents that can affect membrane potential of a target cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. § 120 to and isa divisional of U.S. Application, U.S. Ser. No. 14/739,908, filed Jun.15, 2015, which claims priority under 35 U.S.C. § 120 to and is acontinuation of U.S. Application, U.S. Ser. No. 13/818,432, filed May13, 2013, which is a national stage filing under 35 U.S.C. § 371 ofinternational PCT application, PCT/US2011/048793, filed Aug. 23, 2011,which claims benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplications, U.S. Ser. No. 61/412,972, filed Nov. 12, 2010, and U.S.Ser. No. 61/376,049, filed Aug. 23, 2010. U.S. Provisional Applications,U.S. Ser. No. 61/412,972 filed Nov. 12, 2010, and U.S. Ser. No.61/376,049, filed Aug. 23, 2010 are herein incorporated into thisapplication by reference in their entirety.

FIELD OF THE INVENTION

The field of the invention relates to methods, constructs, andcompositions for optically measuring electrical potential across aphospholipid bilayer.

BACKGROUND

Membrane-enclosed biological structures can support a voltage differencebetween the inside and the outside of the membrane. This voltage, alsocalled membrane potential, serves a variety of biological functions,including carrying information (e.g., in neurons), acting as anintermediate in production of ATP (e.g., in bacteria and mitochondria),powering the flagellar motor (e.g., in bacteria), and controllingtransport of nutrients, toxins, and signaling molecules across the cellmembrane (in bacteria and eukaryotic cells).

In spite of its fundamental biological role, membrane potential is verydifficult to measure. Electrophysiology involves positioning electrodeson both sides of the membrane to record voltage directly.Electrophysiological experiments are slow to set up, can only beperformed on one or a few cells at a time, cannot access deeply buriedtissues (e.g., in vivo), do not work for cells that are too small (e.g.bacteria) or are enclosed in a hard cell wall (e.g. yeast), or aremotile (e.g., sperm) cannot be applied to long-term measurements, andusually damage or kill the cell under study.

Accordingly, novel methods for measuring membrane potential are needed.

SUMMARY OF THE INVENTION

Described herein are methods of harnessing microbial rhodopsins asoptical sensors to detect voltage across phospholipid layers. We havediscovered thatour novel system allows us to optically measure membranepotential of a cell or membrane bound cellular compartment, such asintracellular organelles and artificial cells or other lipid membranebound structures.

The methods comprise expressing a microbial rhodopsin in the cell orcellular organelle, exposing the cell to a light source and detectingthe emitted fluorescence from the microbial rhodopsin, wherein theintensity of the emitted fluorescence reflects membrane potential. Themethod allows measurement of membrane potential without the use ofelectrodes. The method further allows monitoring membrane potentialchanges in response to external stimulus or stimuli. This is importantnot only for research but also, for example, if one wants to screencandidate agents for their capacity to affect membrane potential, e.g.,in drug screens.

Also provided are cells expressing microbial rhodopsins and modifiedmicrobial rhodopsins as well as nucleic acids constructs encodingmodified archaerhodopsins useful in measuring membrane potential andchanges thereof in eukaryotic cells. In some embodiments, the opticalsensors described herein have their endogenous ion pump activity reducedor inhibited partially or substantially completely compared to thenative microbial rhodopsin protein. This permits the optical sensors tosense voltage but not to participate in altering voltage throughestablishing ionic gradients. The detection of the voltage and itschanges can be visualized and measured using optical systems.

The present invention is based, at least in part, on the discovery thatmicrobial rhodopsin proteins, such as archaerhodopsins orproteorhodopsins and modified versions thereof having reduced ionpumping activity (compared to the natural microbial rhodopsin proteinfrom which it is derived) can be used as optical sensors to sensevoltage across membranes in a cell. That is, the microbial rhodopsin andthe modified microbial rhodopsin proteins can be used to measuremembrane potential of a cell and changes in the membrane potential. Theconstructs and methods can also be used for in vivo imaging of organsand organisms, such as a zebrafish, that could not be studied due toelectrode size constraints. This is important not only in research butalso for screening novel candidate agents for their capacity to affectmembrane potential in cells.

We have developed Proteorhodopsin Optical Proton Sensor (PROPS), whichfunction primarily in bacterial cells, and a family ofarchaerhodopsin-based fluorescent voltage-indicating proteins (VIPs)that also function in mammalian cells, including neurons and human stemcell-derived cardiomyocytes. The VIPs are based on voltage indicatorsderived from Archaerhodopsin 3 (Arch) and its homologues. These proteinsindicate electrical dynamics with sub-millisecond temporal resolutionand sub-micron spatial resolution. Using VIPs, we demonstratednon-contact, high-throughput, and high-content methods for measuring byusing optical detection of electrical dynamics in mammalian cells andtissues.

The optical sensors described herein are not constrained by the need forelectrodes and permit electrophysiological studies to be performed ine.g., subcellular compartments (e.g., mitochondria) or in small cells(e.g., bacteria). The optical sensors described herein can be used indrug screening, research settings, and for in vitro and in vivo imagingof voltage changes in both eukaryotic and prokaryotic cells.

We describe voltage indicator proteins and constructs expressing suchproteins. The constructs have optionally a cell-type specific promoterthat turns on when the cells are differentiated, e.g., to neuronalcells, such as neurons, or to cardiac cells, such as cardiomyocytes,purkinje cells, or sinusoidal cells. The constructs may further includetargeting signals such as mitochondrial targeting signals to direct thevoltage indicator protein to a desired membrane location. We providecells and cell lines transiently and stably expressing these proteins,including human stem cells, such as induced pluripotent cells (iPSC) orembryonal stem cells (ESC), neural progenitor cells and neural cells,and cardiac progenitor cells and cardiac myocytes. We also describemethods for screening drugs using the described voltage indicatorproteins.

The cells, whether they be prokaryotic or eukaryotic cells, used in themethods of the invention are typically engineered to express themicrobial rhodopsin or the modified microbial rhodopsin as they do notnaturally express the microbial rhodopsin protein that is used in themethods of the invention.

Accordingly, in one embodiment, the invention provides a method formeasuring membrane potential in a cell expressing a nucleic acidencoding a microbial rhodopsin protein, the method comprising the stepsof (a) exciting, in vitro, ex vivo or in vivo, at least one cellcomprising a nucleic acid encoding a microbial rhodopsin protein withlight of at least one wave length; and (b) detecting, in vitro, ex vivoor in vivo, at least one optical signal from the at least one cell,wherein the level of fluorescence emitted by the at least one cellcompared to a reference is indicative of the membrane potential of thecell.

In some aspects of any embodiment or aspect of the invention, themicrobial rhodopsin protein is a modified microbial rhodopsin proteinwith reduced ion pumping activity compared to a natural microbialrhodopsin protein from which it is derived.

In some aspects of any embodiment or aspect of the invention, themicrobial rhodopsin protein is a member of a proteorhodopsin family ofproteins.

In some aspects of any embodiment or aspect of the invention, themicrobial rhodopsin protein is a member of an archaerhodopsin family ofproteins.

In some aspects of any embodiment or aspect of the invention, the atleast one wave length is a wave length between λ=594-645. Range of wavelength between λ=630-645 nm can also be used.

In some aspects of any embodiment or aspect of the invention, the cellis a prokaryotic cell. The prokaryotic cell can be Gram negative or Grampositive. The prokaryotic cell can be pathogenic or non-pathogenic.

In some aspects of any embodiment or aspect of the invention, the cellis a eukaryotic cell.

In some aspects of any embodiment or aspect of the invention, theeukaryotic cell is a mammalian cell.

In some aspects of any embodiment or aspect of the invention, theeukaryotic cell is a stem cell or a pluripotent or a progenitor cell.

In some aspects of any embodiment or aspect of the invention, theeukaryotic cell is an induced pluripotent cell.

In some aspects of any embodiment or aspect of the invention, theeukaryotic cell is a neural cell.

In some aspects of any embodiment or aspect of the invention, theeukaryotic cell is a cardiomyocyte.

The cells can be cultured in vitro, ex vivo or the cells can be part ofan organ or organism. Exemplary cells include bacteria, yeast, a plantcell, and a cell from vertebrate and non-vertebrate animal. In someembodiments, the eukaryotic cells are human cells. In some embodiments,the eukaryotic cells are non-human cells. In some embodiments, the cellsdo not naturally express the microbial proteorhodopsin used in themethods.

In some aspects of any embodiment or aspect of the invention, the methodfurther comprises a step of transfecting, in vitro, ex vivo or in vivo,the at least one cell with a vector comprising the nucleic acid encodingthe microbial rhodopsin protein. The cells can be transfectedtransiently or stably.

In some aspects of any embodiment or aspect of the invention, thenucleic acid encoding the microbial rhodopsin protein is operably linkedto a cell-type specific promoter.

In some aspects of any embodiment or aspect of the invention, thenucleic acid encoding the microbial rhodopsin protein is operably linkedto a membrane-targeting sequence.

In some aspects of any embodiment or aspect of the invention, themembrane-targeting sequence is a plasma membrane targeting sequence.

In some aspects of any embodiment or aspect of the invention, themembrane-targeting sequence is a subcellular compartment-targetingsequence.

In some aspects of any embodiment or aspect of the invention, thesubcellular compartment is selected from a mitochondrial membrane, anendoplasmic reticulum, a sarcoplastic reticulum, a synaptic vesicle, anendosome and a phagosome.

In some aspects of any embodiment or aspect of the invention, themicrobial rhodopsin gene is operably linked to a nucleic acid encodingan additional fluorescent protein or a chromophore.

In some aspects of any embodiment or aspect of the invention, the atleast one additional fluorescent protein is a protein capable forindicating ion concentration in the cell.

In some aspects of any embodiment or aspect of the invention, the atleast one additional fluorescent protein capable for indicating ionconcentration is a calcium indicator.

In some aspects of any embodiment or aspect of the invention, thefluorescent protein capable for indicating ion concentration is a pHindicator.

In some aspects of any embodiment or aspect of the invention, thefluorescent protein is capable of undergoing nonradiative fluorescenceresonance energy transfer to the microbial rhodopsin, with a rate ofenergy transfer dependent on the membrane potential.

In some aspects of any embodiment or aspect of the invention, brightnessof the fluorescent protein is insensitive to membrane potential andlocal chemical environment, and thereby serves as a reference againstwhich to compare the fluorescence of the microbial rhodopsin

In some aspects of any embodiment or aspect of the invention, the methodfurther comprises steps of exciting, in vitro, ex vivo or in vivo, theat least one cell with light of at least a first and a secondwavelength; and detecting, in vitro, ex vivo, or in vivo, the at leastfirst and the second optical signal resulting from the excitation withthe at least the first and the second wavelength, which is differentfrom the at least first wave length, from the at least one cell.

In some aspects of any embodiment or aspect of the invention, the atleast second wave length is between λ=447-594 nm.

In some aspects of any embodiment or aspect of the invention, the methodfurther comprises a step of calculating the ratio of the fluorescenceemission from the microbial rhodopsin to the fluorescence emission ofthe fluorescent protein to obtain a measurement of membrane potentialindependent of variations in expression level.

In some aspects of any embodiment or aspect of the invention, the methodfurther comprises the step of exposing, in vitro, ex vivo, or in vivo,the at least one cell to a stimulus capable of or suspected to becapable of changing membrane potential.

In some aspects of any embodiment or aspect of the invention, thestimulus a candidate agent. In some embodiments, at least one candidateagent is administered. In some embodiments a combination of at least twocandidate agents are administered simultaneously or in series.

In some aspects of any embodiment or aspect of the invention, thestimulus is a change to the composition of the cell culture medium.

In some aspects of any embodiment or aspect of the invention, thestimulus is an electrical current.

In some aspects of any embodiment or aspect of the invention, the methodfurther comprises the step of measuring, in vitro, ex vivo or in vivo,the at least one optical signal at least at a first and at least at asecond time point.

In some aspects of any embodiment or aspect of the invention, the atleast first time point is before exposing the at least one cell to astimulus and the at least second time point is after exposing the atleast one cell to the stimulus.

In some aspects of any embodiment or aspect of the invention, the methodfurther comprises a step of measuring the ratio of fluorescence betweenthe optical signals from the exposure to the at least first wave lengthand the at least second wave length.

In some aspects of any embodiment or aspect of the invention, the methodcomprises use of a plurality of cells. For example in a high-throughputassay format. For example, in such embodiments, a plurality of cellsexpressing the microbial rhodopsin proteins can be exposed to a numberof candidate agents, such as drug candidates, and screened for thecandidate agents' ability to affect the membrane potential of the cell.

In some aspects of any embodiment or aspect of the invention, theeukaryotic cell is a human cell. In some embodiments, the eukaryoticcell is a non-human cell.

In another embodiment, the invention provides an isolated and purifiednucleic acid encoding a modified member of an archaerhodopsin family ofproteins with reduced ion pumping activity compared to a natural memberof an archaerhodopsin family of proteins from which it is derived.

In some aspects of any embodiment or aspect of the invention, themodified member of an archaerhodopsin family of proteins with reducedion pumping activity compared to a natural member of an archaerhodopsinfamily of proteins from which it is derived comprises a mutated protonacceptor proximal to the Schiff Base.

In some aspects of any embodiment or aspect of the invention, theisolated and purified nucleic acid is operably linked to a nucleic acidencoding a membrane-targeting sequence.

In some aspects of any embodiment or aspect of the invention, themembrane-targeting sequence is a subcellular membrane-targetingsequence.

In some aspects of any embodiment or aspect of the invention, thesubcellular membrane is a mitochondrial membrane, an endoplasmicreticulum, a sarcoplastic reticulum, a synaptic vesicle, an endosome ora phagosome.

In some aspects of any embodiment or aspect of the invention, theisolated and purified nucleic acid is operably linked to a cell-typespecific promoter.

In some aspects of any embodiment or aspect of the invention, theisolated and purified nucleic acid is operably linked to at least onenucleic acid encoding an additional fluorescent protein or achromophore.

In some aspects of any embodiment or aspect of the invention, theadditional fluorescent protein is green fluorescent protein or a homologthereof.

In some aspects of any embodiment or aspect of the invention, theisolated and purified nucleic acid is operably linked to a nucleic acidencoding a fluorescent protein capable of undergoing fluorescenceresonance energy transfer to the microbial rhodopsin, with the rate ofenergy transfer dependent on the membrane potential.

In some aspects of any embodiment or aspect of the invention, theisolated and purified nucleic acid further comprises a vector.

In some aspects of any embodiment or aspect of the invention, the vectoris a viral vector, such as a lentiviral vector or an adeno-associatedvirus (AAV) vector.

In another embodiment, the invention provides a kit comprising theisolated and purified nucleic acid as described above. The nucleic acidmay be provided in a buffer solution or in a dried, such as lyophilizedform in a suitable container. In some embodiments the kit furthercomprises buffers and solutions for performing the methods or the assaysof the invention. Based on the description provided in thespecification, a skilled artisan will be able to pick and choose theappropriate reagents for such a kit. The kit may comprise one or moretransfection agents, one or more buffers, one or more cell culturemedia, and one or more containers, such as cell culture plates orarrays, to perform the methods and assays of the invention. Instructionmanuals comprising instructions for performing the assay may also beincluded with the kits.

In another embodiment, the invention provides an isolated cellcomprising a nucleic acid encoding a microbial rhodopsin protein. Thecell is typically engineered to express the microbial rhodopsin protein.

In some aspects of any embodiment or aspect of the invention, themicrobial rhodopsin protein is a modified microbial rhodopsin proteinwith reduced ion pumping activity compared to a natural microbialrhodopsin protein from which it is derived.

In some aspects of any embodiment or aspect of the invention, themodified microbial rhodopsin protein comprises a mutated proton acceptorproximal to the Schiff Base.

In some aspects of any embodiment or aspect of the invention, themicrobial rhodopsin is a member of a proteorhodopsin family.

In some aspects of any embodiment or aspect of the invention, themicrobial rhodopsin is a member of a archaerhodopsin family.

In some aspects of any embodiment or aspect of the invention, the cellis a eukaryotic cell.

In some aspects of any embodiment or aspect of the invention, the cellis a prokaryotic cell. In some embodiments, the prokaryotic cell is aGram positive cell. In some embodiments, the prokaryotic cell is a Gramnegative cell. In some embodiments, the prokaryotic cell is a pathogeniccell.

In some aspects of any embodiment or aspect of the invention, themodified microbial rhodopsin gene is operably linked to a promoter.

In some aspects of any embodiment or aspect of the invention, thepromoter is a cell-type specific promoter.

In some aspects of any embodiment or aspect of the invention, thenucleic acid encoding the modified microbial rhodopsin protein isoperably linked to a membrane-targeting nucleic acid.

In some aspects of any embodiment or aspect of the invention, thenucleic acid encoding the modified microbial rhodopsin protein isoperably linked to a nucleic acid encoding at least one additionalfluorescent protein or a chromophore.

In some aspects of any embodiment or aspect of the invention, at leastone additional fluorescent protein is a green fluorescent protein or ahomolog thereof.

In some aspects of any embodiment or aspect of the invention, the atleast one additional fluorescent protein is a fluorescent proteincapable of undergoing fluorescence resonance energy transfer to themicrobial rhodopsin, with a rate of energy transfer dependent on themembrane potential.

In some aspects of any embodiment or aspect of the invention, the atleast one additional fluorescent protein is a fluorescent protein whosebrightness is insensitive to membrane potential and local chemicalenvironment

In some aspects of any embodiment or aspect of the invention, the cellfurther comprises a nucleic acid encoding a fluorescent protein capableof indicating ion concentration in the cell.

In some aspects of any embodiment or aspect of the invention, the cellis a stem cell, a pluripotent cell, or an induced pluripotent cell, ordifferentiated or undifferentiated progeny thereof.

In some aspects of any embodiment or aspect of the invention, thedifferentiated cell is a neuron.

In some aspects of any embodiment or aspect of the invention, thedifferentiated cell is a cardiomyocyte.

In one embodiment, the invention provides a kit comprising a cell or aplurality of cells of as described above in a suitable cell culturemedium and a container. The kit may comprise frozen cells in a suitablemedium. Other reagents, such as one or more buffers, one or more cellculture media, and one or more containers may be included in the kit.The kit may also include instructions for the methods of using the cellsin the methods as described herein.

In another embodiment, the invention provides a method of making anengineered cell for optical measurement of membrane potential comprisingthe steps of transfecting a cell with a nucleic acid encoding amicrobial rhodopsin protein. The transfection may be a transienttransfection or a stable transfection.

In some embodiments, the cell is a prokaryotic cell. The prokaryoticcell may be Gram positive or Gram negative. In some aspects, theprokaryotic cell is a pathogenic bacterium. The cell can also be a stemcell, a pluripotent cell, a differentiated cell or an immortalized cellor a cell line. The cell can be an isolated cell or a part of an organor an organism, such as a zebrafish or a non-human embryo or a humanembryo.

In one aspect of this embodiment, and any aspect of this embodiment, themicrobial rhodopsin protein is a modified microbial rhodopsin protein.

In one aspect of this embodiment, and any aspect of this embodiment, thenucleic acid encoding the microbial rhodopsin protein is operably linkedto the differentiated cell type-specific promoter.

In one aspect of this embodiment, and any aspect of this embodiment, thenucleic acid encoding the microbial rhodopsin protein is operably linkedto at least one additional gene encoding a fluorescent protein or achromophore.

In one aspect of this embodiment, the at least one additional geneencoding a fluorescent protein is a green fluorescent protein or ahomolog thereof.

In one aspect of this embodiment, and any aspect of this embodiment, thenucleic acid encoding the microbial rhodopsin protein is operably linkedto a fluorescent protein capable of indicating ion concentration in thecell.

Exemplary nucleic acids and nucleic acid constructs are providedthroughout this specification and their nucleic acid sequences areprovided in the accompanying Sequence Listing. Typically, the modifiedmicrobial rhodopsins are modified to at least reduce the ion pumpingactivity of such proteins by mutating the proton acceptor proximal tothe Schiff Base. However, the invention is not intended to be limited tothese examples, as similar optical measurement is possible for anynumber of the existing microbial rhodopsin proteins. Any such proteinmay be used in the methods of the invention and any such protein mayalso be modified to reduce its ion pumping activity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a mechanism of voltage sensitivity in the D97N mutant ofGreen Proteorhodopsin. Left: Green Proteorhodopsin (a) spans a lipidbilayer membrane (b). Right: Close-up showing a chromophore, retinal(c), covalently linked to the protein backbone via a Schiff Base (d).Aspartic acid 97 in the wild-type structure has been mutated toasparagine (e) to decrease the pKa of the Schiff Base from the wild-typevalue of >12 to the value 9.8 and to eliminate the proton-pumpingphotocycle. A change in the voltage drop across the membrane changes thelocal electrochemical potential for a proton (f) to reside on the SchiffBase, and thereby changes the acid-base equilibrium. The absorptionspectrum and fluorescence of the retinal depend on the state ofprotonation of the Schiff Base: the protonated form is fluorescent, thedeprotonated form is not. The voltage across the membrane is determinedby measuring the fluorescence.

FIGS. 2A-2D show that GPR D97N is a transmembrane protein that showshighly photostable and environmentally sensitive fluorescence. E. Colicells expressing GPR D97N were excited at a wavelength of 633 nm andimaged via fluorescence emission of GPR D97N between 660-760 nm. Theprotein is localized in the cell periphery as expected for atransmembrane protein; FIG. 2A shows visible absorption spectra of GPRD97N in whole E. coli at pH 7 and pH 11; FIG. 2B shows fluorescenceemission spectra of purified GPR D97N protein solubulized inoctyl-glucoside at pH 7 and pH 11; FIG. 2C shows photobleaching curvesof GPR D97N and the organic dye Alexa 647 (Molecular Probes) underidentical illumination conditions. GPR D97N is more photostable than anyother known fluorescent protein under comparable illuminationintensities. FIG. 2D shows pH titration of the Schiff base as monitoredby visible absorption, in the wild-type and D97N mutants of GPR. The pKaof the D97N mutant is 9.8 and the pKa of the wild-type protein is >12.

FIG. 3 shows fluorescence brightness of GPR D97N as a function of pH invivo and in vitro. In both cases the fluorescence decreases at high pHdue to the deprotonation of the Schiff Base. The pKa in E. coli ismeasured in cells whose membrane has been made permeable to protons viaaddition of Carbonyl cyanide 3-chlorophenylhydrazone (CCCP). Thistreatment is necessary because the proton binds to the Schiff Base fromthe cytoplasmic side and in the absence of CCCP the cells maintain anearly constant cytoplasmic pH in the presence of swings in the externalpH. The pKa in the cells and in the purified protein differ due to thelocal environmental effects in the cell.

FIGS. 4A and 4B show fluorescence blinking in E. coli expressing GPRD97N. FIG. 4A shows a film strip of three E. coli at pH 7.5. Eachexposure is 100 ms. The brightness of individual cells varies with time.FIG. 4B shows a blinking pattern from a single cell at pH 7.5. Eachtrace is a 50 second (s) record of the intensity. The time after thestart of the experiment is indicated on the right. Cells continued toblink throughout the 1 hour experiment. The same cell shows fast blinks(0 and 7 minutes), slow blinks (28 minutes), and ‘ringing’ behavior (18minutes).

FIG. 5 shows a map of the plasmid containing Archaerhodopsin 3 (Arch3,also referred to in some instances herein as Ar-3) as described in thesyntheticbiology web site (world wide web atsyntheticneurobiology.org/protocols/protocoldetail/36/10).

FIGS. 6A-6E show that Arch is a fluorescent voltage indicator. FIG. 6Ashows a model of Arch as a voltage sensor. pH and membrane potential canboth alter the protonation of the Schiff base. The crystal structureshown is bacteriorhodopsin; the structure of Arch has not been solved.

FIG. 6B shows absorption (solid line) and fluorescence emission (Em,see, dashed line) spectra of purified Arch at neutral and high pH. FIG.6C top shows a HEK cell expressing Arch, visualized via Archfluorescence. FIG. 6C bottom shows a pixel-weight matrix regions ofvoltage-dependent fluorescence. Scale bar 10 μm. FIG. 6D showsfluorescence of Arch as a function of membrane potential. Thefluorescence was divided by its value at −150 mV. FIG. 6E shows dynamicresponse of Arch to steps in membrane potential between −70 mV and +30mV. The overshoots on the rising and falling edges were an artifact ofelectronic compensation circuitry. Data were an average of 20 cycles.Inset shows that step response occurred in less than the 0.5 msresolution of the imaging system.

FIGS. 7A-7D show optical recording of action potentials with Arch3 WT.Cultured rat hippocampal neuron expressing Arch-GFP were imaged viafluorescence of GFP. Arch fluorescence was shown in cyan and regions ofvoltage-dependent fluorescence were shown in red. FIG. 7A showswhole-cell membrane potential determined via direct voltage recording(bottom, dotted line) and weighted Arch3 fluorescence (top, solid line)during a single-trial recording of a train of action potentials. Insetshows an averaged spike response for 269 events in a single cell,showing voltage (dotted line) and fluorescence (solid line). FIG. 7Bshows recording of multiple spike-trains from a single cell. Currentinjections (shown in black dotted line) of 200 pA were applied to aneuron expressing Arch WT. Action potentials (shown in grey line) werereadily detected via fluorescence over multiple rounds of currentinjection. FIG. 7C shows sub-cellular localization of an actionpotential. We took an image of a field of neuronal processes expressingArch and created a weight matrix indicating pixels, shown as images inthe Figure, whose fluorescence co-varied with the recorded potential inred, overlaid on the time-average Arch fluorescence which was shown incyan. We detected sub-cellular regions within the electrically activecell. The figure shows a timecourse of an action potential determinedvia fluorescence (F) on the top graph (averaged over n=100 spikes)corresponding to each of the regions indicated. Also shown is theelectrical recording of the action potential (V, bottom graph). FIG. 7Dshows heterogeneous dynamics of an action potential within a singleneuron, computed from an average of n=33 spikes. The region indicated bythe arrow in the pixel map (see also graph) lags behind the rest of thecell by ˜1 ms (black arrows). Scale bar 5 μm.

FIGS. 8A-8D demonstrate that Arch D95N shows voltage-dependentfluorescence but no photocurrent. FIG. 8A shows photocurrents in Arch 3WT and Arch 3 D95N mutant, expressed in HEK cells clamped at V=0. Cellswere illuminated with pulses of light at λ=640 nm, 1800 W/cm². FIG. 8Bshows that Arch D95N fluorescence increased 3-fold between −150 mV and+150 mV, with nearly linear sensitivity from −120 to +120 mV. Insetshows a map of voltage sensitivity. Scale bar 5 inn. FIG. 8C shows thatthe step response comprised a component faster than 500 μs (20% of theresponse) and a component with a time constant of 41 ms. FIG. 8D showsthat Arch D95N provided highly accurate estimates of membrane potential,clearly resolving voltage steps of 10 mV, with a noise in the voltageestimated from fluorescence of 260 μV/(Hz)^(1/2) over timescales<12 s.

FIGS. 9A-9C show optical recording of action potentials with ArchD95N.FIG. 9A shows electrically recorded membrane potential of a neuronexpressing Arch WT, subjected to pulses of current injection and laserillumination (I=1800 W/cm2, λ=640 nm). Illumination generated sufficientphotocurrent to suppress action potentials when the cell was nearthreshold. Grey bars indicate laser illumination. FIG. 9B is same asFIG. 9A in a neuron expressing Arch D95N, showing no effect ofillumination on spiking or resting potential. We showed a neuronexpressing Arch D95N, showing Arch D95N fluorescence (shows in cyan inthe experiment), and regions of voltage-dependent fluorescence (shown inred in the experiment). FIG. 9C shows whole-cell membrane potentialdetermined via electrical recording (bottom, voltage line) and weightedArchD95N fluorescence (top, fluorescence line) during a single-trialrecording of a train of action potentials.

FIG. 10 shows existing genetically encoded fluorescent voltageindicators classified according to their sensitivity and speed—the twokey parameters that determine the performance of an indicator. VSFPs,FLARE and SPARC represent indicators based on fusions of GFP homologuesto membrane proteins. The exemplary proteins we have developed are theProteorhodopsin Optical Proton Sensor (PROPS), Arch3 WT, and Arch3 D95N,shown on the upper right. PROPS functions in bacteria, while Arch3 WTand Arch3 D95N function in mammalian cells. Note the logarithmic axes.Microbial rhodopsin-based voltage indicators are much faster and farmore sensitive than other indicators.

FIGS. 11A-11D show optical recordings of action potentials in a singleHL-1 mouse cardiomyocyte expressing Arch3 D95N-eGFP. Action potentialswere recorded for up to 1000 s, with no signs of phototoxicity. Thisexperiment is the first quantitative measurement of cardiac actionpotentials with a genetically encoded voltage indicator. We showed anoverlay showing fluorescence of Arch D95N and GFP in a Arch D95N-GFPfusion. FIG. 11A shows a comparison of the action potential determinedfrom patch clamp recording (dashed line) and fluorescence (solid line).

FIGS. 11B-11D show optical recordings of the action potentials in asingle HL-1 cell over increasingly long intervals. Data in 11D have beencorrected for photobleaching.

FIG. 12 shows optical recordings of action potentials in human inducedpluripotent stem cell (iPS)-derived cardiomyocytes, expressing Arch3D95N-eGFP. Human induced pluripotent stem cells (hiPSC) were provided byCellular Dynamics Inc. Cells were plated into MatTek dishes coated in0.1% gelatin at a density of 20, 50, or 75 thousand cells per squarecentimeter. These conditions showed cells that were sparse and did notbeat spontaneously (20K), a confluent monolayer that did beatspontaneously (50K), and a dense monolayer (75K). iPS cells were platedand maintained for 48 hours in plating medium, and thereafter fed every48 hours with maintenance medium (both from Cellular Dynamics). iPScells were transfected using Mirus LT-1 according to the manufacturerdirections. To a tube containing 20 uL of OPTI-MEM®, we added 200 ng ofDNA and 1.2 uL of the LT-1 transfection reagent. The DNA mixture wasincubated at room temperature for 20 minutes. Fresh maintenance mediumwas added to the iPS cells during the incubation and the DNA mixture wasadded dropwise over the plate. Cells were imaged 48 to 96 hours aftertransfection. We observed synchronized beating of adjoining cells,indicating that VIPs can probe intercellular conduction. We recorded formore than 10 minutes continuously, with little phototoxicity. Within thepopulation of cells we observed cells with action potentials matchingthose of ventricular, atrial, and nodal cells, as expected for thispopulation. Addition of drugs led to changes in the action potentialwaveform that matched changes reported by conventional patch clamp. Cell1 fluorescence information is indicated as a solid line and Cell 2fluorescence as a dashed line on the photos fluorescence vs. time.

FIG. 13 illustrates constructs of fused VIPs with GFP-homologue proteinsto develop a series of improved voltage indicators. The length of eachbar indicates the length of the protein sequence or linker region. Thecolor indicates the color of the fluorescence of the correspondingprotein. All constructs were constructed with both Arch WT and Arch D95Nbackbones. The sequences for the constructs are provided in the SequenceListing as follows:

pADD247/248 SEQ ID NO: 48 pADD286/287 SEQ ID NO: 53 pADD292/293 SEQ IDNO: 50 pADD294 (D95N only) SEQ ID NO: 54 pADD297/300 SEQ ID NO: 51pADD259/298 SEQ ID NOS: 55 and 52, respectively Pfck:Arch3(WT/D95N)-EGFPSEQ ID NOS: 56-57, respectively pADD269/270 SEQ ID NO: 49Pcmv:Arch3(WT/D95N)-GCaMP SEQ ID NOS: 58-59, respectively

FIGS. 14A-14B illustrate mechanism of ssFRET. FIG. 14A shows that whenthe Schiff Base (SB) on the retinal is protonated, the absorptionspectrum of the retinal (solid) overlaps with the emission spectrum ofthe GFP (dotted), and the fluorescence of the GFP is quenched. However,the retinal itself is fluorescent in this state. FIG. 14B shows thatwhen the SB is deprotonated, the GFP fluorescence (solid line) becomesde-quenched and the retinal fluorescence vanishes.

FIG. 15 shows distance dependence of ssFRET signal. As the distancebetween the mOrange2 and the Arch chromophores decreased, the magnitudeof the ssFRET signal increased.

FIG. 16 shows a step response of Arch 3 fluorescence and mOrangefluorescence in pADD294. Here the mOrange2 signal has been inverted tofacilitate comparison with the Arch 3 signal. The similarity of thetimecourses is consistent with modulation of the mOrange2 fluorescencevia ssFRET. The voltage step is from −70 mV to +30 mV.

FIGS. 17A-17C show pH-dependent spectra of Arch 3 WT and D95N. FIG. 17Ashows Arch WT absorption at neutral (bold line) and high (this line) pH.At neutral pH, Arch absorbed maximally at 558 nm. Fluorescence emission(dashed line) was recorded on 2 μM protein solubilized in 1% DM, withλexc=532 nm. FIG. 17B shows Arch D95N spectra under the same conditionsas in FIG. 17A. The absorption maximum was at 585 nm. FIG. 17C showsabsorption spectra that were recorded on purified protein between pH6-11. Singular Value Decomposition of absorption spectra between 400-750nm was used to calculate the fraction of the SB in the protonated stateas a function of pH. The result was fit to a Hill function to determinethe pKa of the SB.

FIG. 18 shows frequency response of Arch 3 WT. A chirped sine wave withamplitude 50 mV and frequency from 1 Hz-1 kHz was applied to a HEK cellexpressing Arch 3 WT (wild type). Membrane potential {circumflex over(V)}_(FL) was determined from fluorescence and the Fourier transform of{circumflex over (V)}_(FL) was calculated. The uptick at 1 kHz is anartifact of electronic compensation circuitry. Inset: power spectrum ofnoise in {circumflex over (V)}_(FL), under voltage clamp at constant V=0mV shows a shot-noise limited noise floor of 470 μV/(Hz)1/2 atfrequencies above 10 Hz. The noise figures reported here are specific toour imaging system and serve primarily as an indicator of the possiblesensitivity of Arch 3.

FIG. 19 shows sensitivity of Arch 3 WT to voltage steps of 10 mV.Whole-cell membrane potential determined via direct voltage recording,V, (bolded black line, showing step-like line on the graph) and weightedArch 3 fluorescence, {circumflex over (V)}₁, (solid narrower lineshowing serrations on the graph).

FIG. 20 shows that arch reports action potentials without exogenousretinal. We made an image of 14 day in vitro (DIV) hippocampal neuronimaged via Arch 3 fluorescence with no exogenous retinal. Electrical(bolded solid black line) and fluorescence (non-bolded line, showingserrated line in the graph) records of membrane potential from theneuron during a current pulse. Action potentials are clearly resolved.

FIG. 21 shows a frequency response of Arch D95N, measured in the samemanner as for Arch 3 WT (FIG. 18).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery thatmicrobial rhodopsin proteins or modified microbial rhodopsin proteinsthat have reduced ion pumping activity, compared to the naturalmicrobial rhodopsin protein from which they are derived, can be used asan optically detectable sensor to sense voltage across membranousstructures, such as in cells and sub-cellular organelles when they arepresent on the cell membrane. That is, the microbial rhodopsin proteinsand the modified microbial rhodopsin proteins can be used to measurechanges in membrane potential of a cell, including prokaryotic andeukaryotic cells. The optical sensors described herein are notconstrained by the need for electrodes and permit electrophysiologicalstudies to be performed in e.g., subcellular compartments (e.g.,mitochondria) or in small cells (e.g., bacteria). The optical sensorsdescribed herein can be used in methods for drug screening, in researchsettings, and in in vivo imaging systems.

Microbial Rhodopsins: Design of Optical Voltage Sensors

Microbial rhodopsins are a large class of proteins characterized byseven transmembrane domains and a retinilydene chromophore bound in theprotein core to a lysine via a Schiff base (Beja, O., et al. Nature 411,786-789 (2001)). Over 5,000 microbial rhodopsins are known, and theseproteins are found in all kingdoms of life. Microbial rhodopsins serve avariety of functions for their hosts: some are light-driven proton pumps(bacteriorhodopsin, proteorhodopsins), others are light-driven ionchannels (channelrhodopsins), chloride pumps (halorhodopsins), or servein a purely photosensory capacity (sensory rhodopsins).

The retinilydene chromophore imbues microbial rhodopsins with unusualoptical properties. The linear and nonlinear responses of the retinalare highly sensitive to interactions with the protein host: smallchanges in the electrostatic environment can lead to large changes inabsorption spectrum. These electro-optical couplings provide the basisfor voltage sensitivity in microbial rhodopsins.

Some of the optical sensors described herein are natural proteinswithout modifications and are used in cells that do not normally expressthe microbial rhodopsin transfected to the cell, such as eukaryoticcells. For example, as shown in the examples, the wild type Arch3 can beused in neural cells to specifically detect membrane potential andchanges thereto.

Some of the microbial rhodopsins are derived from a microbial rhodopsinprotein by modification of the protein to reduce or inhibitlight-induced ion pumping of the rhodopsin protein. Such modificationspermit the modified microbial rhodopsin proteins to sense voltagewithout altering the membrane potential of the cell with its native ionpumping activity and thus altering the voltage of the system. Othermutations impart other advantageous properties to microbial rhodopsinvoltage sensors, including increased fluorescence brightness, improvedphotostability, tuning of the sensitivity and dynamic range of thevoltage response, increased response speed, and tuning of the absorptionand emission spectra.

Mutations that eliminate pumping in microbial rhodopsins in the presentinvention generally comprise mutations to the Schiff base counterion; acarboxylic amino acid (Asp or Glu) conserved on the third transmembranehelix (helix C) of the rhodopsin proteins. The amino acid sequence isRYX(DE) where X is a non-conserved amino acid. Mutations to thecarboxylic residue directly affect the proton conduction pathway,eliminating proton pumping. Most typically the mutation is to Asn orGln, although other mutations are possible, and based on the descriptionprovided herein, one skilled in the art can make different mutants whichalso result in reduced or absent ion pumping by the microbial rhodopsinprotein. In one embodiment, the modified microbial rhodopsin proteins ofthe invention and the methods of the invention comprises the Asp to Asnor Gln mutation, or Glu to Asn or Gln mutation. In some embodiments, theprotein consist essentially of Asp to Asn or Gln mutation, or Glu to Asnor Gln mutation. In some embodiments, the protein consist of Asp to Asnor Gln mutation, or Glu to Asn or Gln mutation.

Provided herein are illustrative exemplary optical voltage sensors anddirections for making and using such sensors. Other sensors that work ina similar manner as optical sensors can be prepared and used based onthe description and the examples provided herein.

Table 1a includes exemplary microbial rhodopsins useful according to thepresent invention. For example, mutations that eliminate pumping inmicrobial rhodopsins in the present invention generally comprisemutations to the Schiff base counterion; a carboxylic amino acid (Asp orGlu) conserved on the third transmembrane helix (helix C) of therhodopsin proteins. Table 1a refers to the amino acid position in thesequence provided as the exemplary Genbank number. However, the positionmay be numbered slightly differently based on the variations in theavailable amino acid sequences. Based on the description of the motifdescribed herein, a skilled artisan will easily be able to make similarmutations into other microbial rhodopsin genes to achieve the samefunctional feature, i.e. reduction in the pumping activity of themicrobial rhodopsin in question.

TABLE 1a Exemplary microbial rhodopsins useful according to the presentinvention. Microbial Rhodopsin Abbreviation Genbank number Amino acidmutation Green-absorbing GPR AF349983; wild-type, D99N (SEQ ID NO: 76)in the proteorhodopsin: a (Nucleotide and protein specification, thismutation is light-driven proton disclosed as SEQ ID also referred to asD97N pump found in NOS 74-75, marine bacteria respectively)Blue-absorbing BPR AF349981; wild-type; D99N (SEQ ID NO: 79)proteorhodopsin: a (Nucleotide and protein light-driven proton disclosedas SEQ ID pump found in NOS 77-78, marine bacteria. respectively)Natronomonas NpSRII Z35086.1; In one D75N (SEQ ID NO: 82) pharaonissensory embodiment only the rhodopsin II: a light- sensory domain, givenactivated signaling by nucleotides 2112-2831 protein found in the ofsequence halophilic bacterium Z35086, is used N. pharaonis. In the(Nucleotide and wild the sensory “sensory domain” domain is paired withprotein disclosed as a transducer domain SEQ ID NOS 80-81, respectively)Bacteriorhodopsin: a BR NC_010364.1, D98N light-driven protonnucleotides 1082241 to (SEQ ID NO: 85) pump found in 1083029, or GenBankHalobacterium sequence M11720.1; salinarum (“M11720.1” nucleotide andprotein disclosed as SEQ ID NOS 83-84, respectively Archaerhodopsin Arch3 (or Ar Chow B. Y. et al., D95N (SEQ ID NO: 87) Arch 3: a light-driven3) Nature 463: 98-102; proton pump found (“Arch 3” wild-type inHalobacterium protein disclosed as sodomense SEQ ID NO: 86)

The following Table 1b includes exemplary additional rhodopsins that canbe mutated as indicated in the methods of the invention:

TABLE 1b Nucleic acid Amino Acid Microbial Rhodopsin AbbreviationGenbank number mutation mutation Fungal Opsin Related Mac AAG01180 (SEQG415 to A D139N (SEQ ID Protein ID NO: 60) NO: 61) Cruxrhodopsin CruxBAA06678 (SEQ G247 to A D83N (SEQ ID ID NO: 62) NO: 63) AlgalBacteriorhodopsin Ace AAY82897 (SEQ G265 to A D89N (SEQ ID ID NO: 64)NO: 65) Archaerhodopsin 1 Ar1 P69051 (SEQ ID G289 to A D97N (SEQ ID NO:66) NO: 67) Archaerhodopsin 2 Ar2 P29563 (SEQ ID G286 to A D96N (SEQ IDNO: 68) NO: 69) Archaerhodopsin 3 Ar3 P96787 (SEQ ID G283 to A D95N (SEQID NO: 70) NO: 71) Archaerhodopsin 4 Ar4 AAG42454 (SEQ G292 to A D98N(SEQ ID ID NO: 72) NO: 73)Voltage Indicating Proteins (VIP)

We have developed a family of fluorescent voltage-indicating proteins(VIPs) based on Achaerhodopsins that function in mammalian cells,including neurons and human stem cell-derived cardiomyocytes. Theseproteins indicate electrical dynamics with sub-millisecond temporalresolution and sub-micron spatial resolution. We have demonstratednon-contact, high-throughput, and high-content studies of electricaldynamics in mammalian cells and tissues using optical measurement ofmembrane potential. These VIPs are broadly useful, particularly ineukaryotic, such as mammalian, including human cells.

We have developed VIPs based on Archaerhodopsin 3 (Arch 3) and itshomologues. Arch 3 is Archaerhodopsin from H. sodomense and it is knownas a genetically-encoded reagent for high-performance yellow/green-lightneural silencing. Gene sequence at GenBank: GU045593.1 (syntheticconstruct Arch 3 gene, complete cds. Submitted Sep. 28, 2009). We haveshown that these proteins localize to the plasma membrane in eukaryoticcells and show voltage-dependent fluorescence.

We have also shown further improved membrane localization, withcomparable voltage sensitivity, in ArchT, gene sequence at GenBank:HM367071.1 (synthetic construct ArchT gene, complete cds. Submitted May27, 2010). ArchT is Archaerhodopsin from Halorubrum sp. TP009:genetically-encoded reagent for high-performance yellow/green-lightneural silencing, 3.5× more light sensitive than Arch 3.

Table 1c summarizes exemplary sequences that can be used to create viralconstructs that express the voltage indicators based on Archaerhodopsin.

TABLE 1c Exemplary sequences that can be used to generate virusconstructs with Arch 3 and ArchT Virus backbone Lentivirus SEQ ID NO: 24Promoter CamKII (neuron specific) SEQ ID NO: 25 CAG enhancer (pancellular) SEQ ID NO: 26 CMV (pan cellular) SEQ ID NO: 27 Ubiquitin (pancellular) SEQ ID NO: 28 Voltage-sensing Arch D95N SEQ ID NO: 29 domainArchT D95N SEQ ID NO: 30

FIG. 10 shows existing genetically encoded fluorescent voltageindicators classified according to their sensitivity and speed—the twokey parameters that determine the performance of an indicator. Theproteins we developed are the Proteorhodopsin Optical Proton Sensor(PROPS), Arch 3 WT, and Arch 3 D95N, shown on the upper right. PROPSonly functions in bacteria, while Arch 3 WT and Arch 3 D95N function inmammalian cells. We showed that microbial rhodopsin-based voltageindicators are faster and far more sensitive than other indicators.

Table 2 shows exemplary approximate characteristics of fluorescentvoltage indicating proteins and contains representative members of allfamilies of fluorescent indicators. Although the list on Table 2 is notcomprehensive, one skilled in the art can readily see thecharacteristics of the types of proteins useful in the presentinvention.

TABLE 2 Representative members of all families of fluorescentindicators. Approx Approx ΔF/F per response Molecule 100 mV timeComments VSFP 2.3, Knopfel, T. 9.5% 78 ms Ratiometric (ΔR/R) et al. J.Neurosci. 30, 14998-15004 (2010) VSFP 2.4 Knopfel, T. et 8.9% 72 msRatiometric (ΔR/R) al. J. Neurosci. 30, 14998-15004 (2010) VSFP 3.1,Lundby, A.,   3% 1-20 ms Protein et al., PLoS One 3, 2514 (2008)Mermaid, Perron, A. et 9.2% 76 Ratiometric (ΔR/R) al. Front MolNeurosci. 2, 1-8 (2009) SPARC, Ataka, K. & 0.5% 0.8 ms ProteinPieribone, V. A. Biophys. J. 82, 509-516 (2002) Flash, Siegel, M. S. &5.1% 2.8-85 ms Protein Isacoff, E. Y. Neuron 19, 735-741 (1997) PROPS,described 150%  5 ms Protein herein; SEQ ID NO: Arch 3 WT, described 66% <0.5 ms Protein herein Arch D95N 100%  41 ms Protein

FIG. 9 shows an optical recording of action potentials in a single rathippocampal neuron. The data represents a single trial, in which spikingwas induced by injection of a current pulse. The fluorescence showsclear bursts accompanying individual action potentials. This experimentis the first robust measurement of action potentials in a singlemammalian neuron using a genetically encoded voltage indicator.

FIG. 11 shows optical recordings of action potentials in a single HL-1mouse cardiomyocyte expressing Arch 3 D95N-eGFP. Action potentials wererecorded for up to 1000 s, with no signs of phototoxicity. Thisexperiment is the first quantitative measurement of cardiac actionpotentials with a genetically encoded voltage indicator.

FIG. 12 shows optical recordings of action potentials in human inducedpluripotent stem cell (iPS)-derived cardiomyocytes, expressing Arch 3D95N-eGFP. Human induced pluripotent stem cells (hiPSC) were provided byCellular Dynamics Inc. Cells were plated into MatTek dishes coated in0.1% gelatin at a density of 20, 50, or 75 thousand cells per squarecentimeter. These conditions showed cells that were sparse and did notbeat spontaneously (20K), a confluent monolayer that did beatspontaneously (50K), and a dense monolayer (75K). iPS cells were platedand maintained for 48 hours in plating medium, and thereafter fed every48 hours with maintenance medium (both from Cellular Dynamics). iPScells were transfected using Mirus LT-1 according to the manufacturerdirections. To a tube containing 20 uL of optimem, we added 200 ng ofDNA and 1.2 uL of the LT-1 transfection reagent. The DNA mixture wasincubated at room temperature for 20 minutes. Fresh maintenance mediumwas added to the iPS cells during the incubation and the DNA mixture wasadded dropwise over the plate. Cells were imaged 48 to 96 hours aftertransfection.

We observed synchronized beating of adjoining cells, indicating thatVIPs can probe intercellular conduction. We recorded for more than 10minutes continuously, with little phototoxicity. Within the populationof cells we observed cells with action potentials matching those ofventricular, atrial, and nodal cells, as expected for this population.Addition of drugs led to changes in the action potential waveform thatmatched changes reported by conventional patch clamp.

Generation of Fusions Between Microbial Rhodopsins and GFP Homologueswith Additional or Improved Properties

We fused VIPs with GFP-homologue proteins to develop a series ofimproved voltage indicators. FIG. 16 illustrates these exemplaryconstructs and FIG. 16 legend provides the sequences for theseconstructs. The new capabilities of these newly developed sensorsinclude, for example, a spectral shift FRET (ssFRET) for enhancedbrightness and 2-photon imaging, ratiometric voltage imaging, andmultimodal sensors for simultaneous measurement of voltage andconcentration.

Spectral Shift FRET (ssFRET) for Enhanced Brightness and 2-photonImaging

A key limitation of the first generation of VIPs was that the endogenousfluorescence of the retinal was dim. Imaging required a specializedsystem comprising an intense red laser, a high numerical apertureobjective, and an electron-multiplying CCD (EMCCD) camera. Ideally onewould like an indicator bright enough to image on a conventionalwide-field or confocal fluorescence microscope, or a 2-photon confocalmicroscope for in vivo applications.

ssFRET provides a path to brighter VIPs as shown in FIG. 10. AGFP-homologue (generically referred to as GFP) is fused to the microbialrhodopsin (see, e.g., FIG. 13). Voltage-dependent changes in theabsorption spectrum of the retinal lead to voltage-dependent rates ofnonradiative fluorescence resonance energy transfer (FRET) between theGFP and the retinal. Retinal in its absorbing, fluorescent statequenches the GFP, while retinal in the non-absorbing, non-fluorescentstate does not quench the GFP. Thus one obtains anti-correlatedfluorescence emission of the GFP and the retinal.

Thus in one embodiment, the invention provides a fusion proteincomprising a GFP that is fused to a microbial rhodopsin or a modifiedmicrobial rhodopsin, such as a proteorhdopsin or archaerhodopsin. Suchfusion proteins can be used in any and all of the methods of the presentinvention.

To maximize the degree of ssFRET between the GFP and the retinal weselected a GFP-homologue, mOrange2, whose emission overlaps maximallywith the absorption of Arch 3 in its protonated state. The rate of FRETfalls off very quickly with increasing distance between chromophores, sowe constructed a series of truncated constructs in which the linker andnon-essential elements of the Arch 3 and mOrange2 were removed. FIG. 15shows that as the distance between the mOrange2 and the Arch 3decreased, the ssFRET signal increased. This same strategy can beapplied to generate ssFRET signals from other microbial rhodopsins andGFP homologues.

We have shown that the time response of fluorescence from mOrange2 to astep in Vm matches the time response of fluorescence from Arch D95N.This observation is consistent with ssFRET. Similar results can be seenfor FIGS. 9A and 11 for fusions to Arch 3 WT.

Ratiometric Voltage Imaging

A key challenge in application of VIPs is to extract accurate values ofthe membrane potential, without systematic artifacts fromphotobleaching, variation in illumination intensity, cell movement, orvariations in protein expression level. In cells that are accessible topatch clamp, one can calibrate the fluorescence as a function ofmembrane potential by varying the membrane potential under externalcontrol. However, a benefit of VIPs is that they function in systemsthat are inaccessible to patch clamp. In these cases direct calibrationis not possible.

The Arch 3 (WT or D95N) fusion with eGFP enables ratiometricdetermination of membrane potential. Similar ratiometric determinationsmay be made using other rhodopsins such as those described in thisapplication using the identical concept. The eGFP fluorescence isindependent of membrane potential, The ratio of Arch 3 fluorescence toeGFP fluorescence provides a measure of membrane potential that isindependent of variations in expression level, illumination, ormovement. This construct does not undergo ssFRET due to the long linkerbetween the eGFP and the Arch 3, and because the emission of eGFP haslittle spectral overlap with the absorption of Arch 3.

Multimodal Sensors for Simultaneous Measurement of Voltage andConcentration

Membrane potential is only one of several mechanisms of signaling withincells. One often wishes to correlate changes in membrane potential withchanges in concentration of other species, such as Ca++, H+ (i.e. pH),Na+, ATP, cAMP. We constructed fusions of Arch with pHluorin (afluorescent pH indicator) and GCaMP3 (a fluorescent Ca++ indicator). Onecan also use fusions with other protein-based fluorescent indicators toenable other forms of multimodal imaging using the concept as taughtherein. Concentration of ions such as sodium, potassium, chloride, andcalcium can be simultaneously measured when the nucleic acid encodingthe microbial rhodopsin is operably linked to or fused with anadditional fluorescent ion sensitive indicator.

Additional Fluorescent Proteins

The term “additional fluorescent molecule” refers to fluorescentproteins other then microbial rhodopsins. Such molecules may include,e.g., green fluorescent proteins and their homologs.

Fluorescent proteins that are not microbial rhodopsins are well knownand commonly used, and examples can be found, e.g., in a review TheFamily of GFP-Like Proteins: Structure, Function, Photophysics andBiosensor Applications. Introduction and Perspective, by Rebekka M.Wachter (Photochemistry and Photobiology Volume 82, Issue 2, pages339-344, March 2006). Also, a review by Nathan C Shaner, Paul ASteinbach, & Roger Y Tsien, entitled A guide to choosing fluorescentproteins (Nature Methods—2, 905-909 (2005)) provides examples ofadditional useful fluorescent proteins.

Targeting VIPs to Intracellular Organelles

We have shown targeting VIPs to intracellular organelles, includingmitochondria, the endoplasmic reticulum, the sarcoplasmic reticulum,synaptic vesicles, and phagosomes. Accordingly, in one embodiment, theinvention provides constructs, such as expression constructs, such asviral constructs comprising a microbial rhodopsin operably linked to asequence targeting the protein to an intracellular organelle, includinga mitochondrium, an endoplasmic reticulum, a sarcoplasmic reticulum, asynaptic vesicle, and a phagosome.

The invention further provides cells expressing the constructs, andfurther methods of measuring membrane potential changes in the cellsexpressing such constructs as well as methods of screening for agentsthat affect the membrane potential of one or more of the intracellularmembranes.

The Key Advantages of Voltage Indicating Proteins (VIPs)

The newly developed VIPs show high sensitivity. In mammalian cells VIPsshow about 3-fold increase in fluorescence between −150 mV and +150 mV.The response is linear over most of this range. We can measure membranevoltage with a precision of <1 mV in a 1 s interval.

The newly developed VIPs show high speed. Arch 3 WT shows 90% of itsstep response in <0.5 ms. A neuronal action potential lasts 1 ms, sothis speed meets the benchmark for imaging electrical activity ofneurons. However, Arch 3 WT retains the photoinduced proton-pumping, soillumination slightly hyperpolarizes the cell.

The modified microbial rhodopsin, Arch 3 D95N, has a 40 ms response timeand lacks photoinduced proton pumping. Although the slower response timeof this construct hampers detection of membrane potential and changesthereto in neurons, the Arch 3 D95N is fast enough to indicate membranepotential and action potentials in other types of cells, for example, incardiomyocytes and does not perturb membrane potential in the cellswherein it is used.

The newly developed VIPs also show high photostability. VIPs arecomparable to GFP in the number of fluorescence photons produced priorto photobleaching. We routinely watch VIPs in mammalian cells for manyminutes, without signs of photobleaching or phototoxicity. VIPs have nohomology to GFP, nor to any other known fluorescent protein.

The newly developed VIPs also show far red spectrum. VIPs are excitedwith a 633 nm laser, and the emission is in the near infrared, peaked at710 nm. The emission is farther to the red than any existing fluorescentprotein. These wavelengths coincide with low cellular autofluorescenceand good transmission through tissue. This feature makes these proteinsparticularly useful in optical measurements of action potential as thespectrum facilitates imaging with high signal-to-noise, as well asmulti-spectral imaging in combination with other fluorescent probes.

The newly developed VIPs further show high targetability. We have imagedVIPs in primary neuronal cultures, cardiomyocytes (HL-1 and humaniPSC-derived), HEK cells, and Gram positive and Gram negative bacteria.We targeted a VIP to the endoplasmic reticulum, and to mitochondria. Theocnstructs are useful also for in vivo imaging in C. elegans, zebrafish,and mice.

With the microbial rhodopsin constructs of the invention furthercomprising a cell type- and/or a time-specific promotors, one can imagemembrane potential in any optically accessible cell type or organelle ina living organism.

In one embodiment, the design of a voltage sensor comprises, consistsof, or consists essentially of selecting at least three elements: apromoter, a microbial rhodopsin voltage sensor, one or more targetingmotifs, and an optional accessory fluorescent protein. Some non-limitingexamples for each of these elements are listed in Tables 1a and 1b, andTable 3 below. In one embodiment, at least one element from each columnis selected to create an optical voltage sensor with the desiredproperties. In some embodiments, methods and compositions for voltagesensing as described herein involves selecting: 1) A microbial rhodopsinprotein, 2) one or more mutations to imbue the protein with sensitivityto voltage or to other quantities of interest and to eliminatelight-driven charge pumping, 3) codon usage appropriate to the hostspecies, 4) a promoter and targeting sequences to express the protein incell types of interest and to target the protein to the sub-cellularstructure of interest, 5) an optional fusion with a conventionalfluorescent protein to provide ratiometric imaging, 6) a chromophore toinsert into the microbial rhodopsin, and 7) an optical imaging scheme.

TABLE 3 Exemplary optical sensor combinations Accessory fluorescentPromoter Voltage sensor Targeting motif protein CMV (SEQ IDhGPR (D97N)(SEQ SS(β2nAChR)(SEQ Venus (SEQ ID NO: 43) NO: 31) ID NO: 35)ID NO: 39) 14x UAS-E1b hGPR (D97N, ±E108Q, SS(PPL) EYFP (SEQ ID NO: 44)(SEQ ID ±E142Q, ±L217D) (SEQ (SEQ ID NO: 40) NO: 32) ID NO: 36)HuC (SEQ ID hBPR (D99N) (SEQ ID ER export motif TagRFP (SEQ ID NO: 45)NO: 33) NO: 37) (SEQ ID NO: 41) ara (SEQ ID hNpSRII (D75N) (SEQTS from Kir2.1 NO: 34) ID NO: 38) (SEQ ID NO: 42) lac MS

In one embodiment, the optical sensor gene is encoded by a deliveryvector. Such vectors include but are not limited to: plasmids (e.g.pBADTOPO, pCI-Neo, pcDNA3.0), cosmids, and viruses (such as alentivirus, an adeno-associated virus, or a baculovirus).

In one embodiment, the green-absorbing proteorhodopsin (GPR) is used asthe starting molecule. This molecule is selected for its relativelyred-shifted absorption spectrum and its ease of expression inheterologous hosts such as E. coli. In another embodiment, theblue-absorbing proteorhodopsin (BPR) is used as an optical sensor ofvoltage. It is contemplated herein that a significant number of themicrobial rhodopsins found in the wild can be engineered as describedherein to serve as optical voltage sensors.

Microbial rhodopsins are sensitive to quantities other than voltage.Mutants of GPR and BPR, as described herein, are also sensitive tointracellular pH. It is also contemplated that mutants of halorhodopsinmay be sensitive to local chloride concentration.

In one embodiment, the voltage sensor is selected from a microbialrhodopsin protein (wild-type or mutant) that provides a voltage-inducedshift in its absorption or fluorescence. The starting sequences fromwhich these constructs can be engineered include, but are not limitedto, sequences listed in Tables 1a-1b, that list the rhodopsin and anexemplary mutation that can be made to the gene to enhance theperformance of the protein product.

Mutations to minimize the light-induced charge-pumping capacity. Theretinal chromophore is linked to a lysine by a Schiff base. A conservedaspartic acid serves as the proton acceptor adjacent to the Schiff base.Mutating this aspartic acid to asparagine suppresses proton pumping.Thus, in some embodiments, the mutations are selected from the groupconsisting of: D97N (green-absorbing proteorhodopsin), D99N(blue-absorbing proteorhodopsin), D75N (sensory rhodopsin II), and D85N(bacteriorhodopsin). In other embodiments, residues that can be mutatedto inhibit pumping include (using bacteriorhodopsin numbering) D96,Y199, and R82, and their homologues in other microbial rhodopsins. Inanother embodiment, residue D95 can be mutated in archaerhodopsin toinhibit proton pumping (e.g., D95N).

Mutations are introduced to shift the absorption and emission spectrainto a desirable range. Residues near the binding pocket can be mutatedsingly or in combination to tune the spectra to a desired absorption andemission wavelength. In bacteriorhodopsin these residues include, butare not limited to, L92, W86, W182, D212, 1119, and M145. Homologousresidues may be mutated in other microbial rhodopsins. Thus, in someembodiments, the mutation to modify the microbial rhodopsin protein isperformed at a residue selected from the group consisting of L92, W86,W182, D212, 1119, M145.

Mutations are introduced to shift the dynamic range of voltagesensitivity into a desired band. Such mutations function by shifting thedistribution of charge in the vicinity of the Schiff base, and therebychanging the voltage needed to add or remove a proton from this group.Voltage-shifting mutations in green-absorbing proteorhodopsin include,but are not limited to, E108Q, E142Q, L217D, either singly or incombination using green-absorbing proteorhodopsin locations as anexample, or a homologous residue in another rhodopsin. In oneembodiment, a D95N mutation is introduced into archaerhodopsin 3 toadjust the pKa of the Schiff base towards a neutral pH.

Optionally mutations are introduced to enhance the brightness andphotostability of the fluorescence. Residues which when mutated mayrestrict the binding pocket to increase fluorescence include (usingbacteriorhodopsin numbering), but are not limited to, Y199, Y57, P49,V213, and V48.

Codon Usage

A large number of mammalian genes, including, for example, murine andhuman genes, have been successfully expressed in various host cells,including bacterial, yeast, insect, plant and mammalian host cells.Nevertheless, despite the burgeoning knowledge of expression systems andrecombinant DNA technology, significant obstacles remain when oneattempts to express a foreign or synthetic gene in a selected host cell.For example, translation of a synthetic gene, even when coupled with astrong promoter, often proceeds much more slowly than would be expected.The same is frequently true of exogenous genes that are foreign to thehost cell. This lower than expected translation efficiency is often dueto the protein coding regions of the gene having a codon usage patternthat does not resemble those of highly expressed genes in the host cell.It is known in this regard that codon utilization is highly biased andvaries considerably in different organisms and that biases in codonusage can alter peptide elongation rates. It is also known that codonusage patterns are related to the relative abundance of tRNAisoacceptors, and that genes encoding proteins of high versus lowabundance show differences in their codon preferences.

Codon-optimization techniques have been developed for improving thetranslational kinetics of translationally inefficient protein codingregions. These techniques are based on the replacement of codons thatare rarely or infrequently used in the host cell with those that arehost-preferred. Codon frequencies can be derived from literature sourcesfor the highly expressed genes of many organisms (see, for example,Nakamura et al., 1996, Nucleic Acids Res 24: 214-215). These frequenciesare generally expressed on an ‘organism-wide average basis’ as thepercentage of occasions that a synonymous codon is used to encode acorresponding amino acid across a collection of protein-encoding genesof that organism, which are preferably highly expressed. In oneembodiment, the codons of a microbial rhodopsin protein are optimizedfor expression in a eukaryotic cell. In one embodiment, the eukaryoticcell is a human cell.

It is preferable but not necessary to replace all the codons of themicrobial polynucleotide with synonymous codons having highertranslational efficiencies in eukaryotic (e.g., human) cells than thefirst codons. Increased expression can be accomplished even with partialreplacement. Typically, the replacement step affects at least about 5%,10%, 15%, 20%, 25%, 30%, more preferably at least about 35%, 40%, 50%,60%, 70% or more of the first codons of the parent polynucleotide.Suitably, the number of, and difference in translational efficiencybetween, the first codons and the synonymous codons are selected suchthat the protein of interest is produced from the syntheticpolynucleotide in the eukaryotic cell at a level which is at least about110%, suitably at least about 150%, preferably at least about 200%, morepreferably at least about 250%, even more preferably at least about300%, even more preferably at least about 350%, even more preferably atleast about 400%, even more preferably at least about 450%, even morepreferably at least about 500%, and still even more preferably at leastabout 1000%, of the level at which the protein is produced from theparent polynucleotide in the eukaryotic cell.

Generally, if a parent polynucleotide has a choice of low andintermediate translationally efficient codons, it is preferable in thefirst instance to replace some, or more preferably all, of the lowtranslationally efficient codons with synonymous codons havingintermediate, or preferably high, translational efficiencies. Typically,replacement of low with intermediate or high translationally efficientcodons results in a substantial increase in production of thepolypeptide from the synthetic polynucleotide so constructed. However,it is also preferable to replace some, or preferably all, of theintermediate translationally efficient codons with high translationallyefficient codons for optimized production of the polypeptide.

Replacement of one codon for another can be achieved using standardmethods known in the art. For example codon modification of a parentpolynucleotide can be effected using several known mutagenesistechniques including, for example, oligonucleotide-directed mutagenesis,mutagenesis with degenerate oligonucleotides, and region-specificmutagenesis. Exemplary in vitro mutagenesis techniques are described forexample in U.S. Pat. Nos. 4,184,917, 4,321,365 and 4,351,901 or in therelevant sections of Ausubel, et al. (CURRENT PROTOCOLS IN MOLECULARBIOLOGY, John Wiley & Sons, Inc. 1997) and of Sambrook, et al.,(MOLECULAR CLONING. A LABORATORY MANUAL, Cold Spring Harbor Press,1989). Instead of in vitro mutagenesis, the synthetic polynucleotide canbe synthesized de novo using readily available machinery as described,for example, in U.S. Pat. No. 4,293,652. However, it should be notedthat the present invention is not dependent on, and not directed to, anyone particular technique for constructing the synthetic polynucleotide.

The genes for microbial rhodopsins (e.g., GPR) express well in E. coli,but less well in eukaryotic hosts. In one embodiment, to enableexpression in eukaryotes a version of the gene with codon usageappropriate to eukaryotic (e.g., human) cells is designed andsynthesized. This procedure can be implemented for any gene usingpublicly available software, such as e.g., the Gene Designer 2.0 package(available on the world wide web at dna20.com/genedesigner2/). Some ofthe “humanized” genes are referred to herein by placing the letter “h”in front of the name, e.g. hGPR. The Arch 3 rhodopsins and mutantsthereof described herein and in the examples are all optimized for humancodon usage.

Applications for VIPs in Screens for Drugs that Target the FollowingTissues or Processes

The constructs disclosed in the present application can be used inmethods for drug screening, e.g., for drugs targeting the nervoussystem. In a culture of cells expressing specific ion channels, one canscreen for agonists or antagonists without the labor of applying patchclamp to cells one at a time. In neuronal cultures one can probe theeffects of drugs on action potential initiation, propagation, andsynaptic transmission. Application in human iPSC-derived neurons willenable studies on genetically determined neurological diseases, as wellas studies on the response to environmental stresses (e.g. anoxia).

Similarly, the optical voltage sensing using the constructs providedherein provides a new and much improved methods to screen for drugs thatmodulate the cardiac action potential and its intercellular propagation.These screens will be useful both for determining safety of candidatedrugs and to identify new cardiac drug leads. Identifying drugs thatinteract with the hERG channel is a particularly promising directionbecause inhibition of hERG is associated with ventricular fibrillationin patients with long QT syndrome. Application in human iPSC-derivedcardiomyocytes will enable studies on genetically determined cardiacconditions, as well as studies on the response to environmental stresses(e.g. anoxia).

Additionally, the constructs of the present invention can be used inmethods to study of development and wound healing. The role ofelectrical signaling in normal and abnormal development, as well astissue repair, is poorly understood. VIPs enable studies of voltagedynamics over long times in developing or healing tissues, organs, andorganisms, and lead to drugs that modulate these dynamics.

In yet another embodiment, the invention provides methods to screen fordrugs that affect membrane potential of mitochondria. Mitochondria playan essential role in ageing, cancer, and neurodegenerative diseases.Currently there is no good probe for mitochondrial membrane potential.VIPs provide such a probe, enabling searches for drugs that modulatemitochondrial activity.

The invention further provides methods to screen for drugs that modulatethe electrophysiology of a wide range of medically, industrially, andenvironmentally significant microorganisms.

Prior to our discovery of VIPs, no measurement of membrane potential hadbeen made in any intact prokaryote. We discovered that bacteria havecomplex electrical dynamics. VIPs enable screens for drugs that modulatethe electrophysiology of a wide range of medically, industrially, andenvironmentally significant microorganisms. For instance, we found thatelectrical activity is correlated with efflux pumping in E. coli.

Changes in membrane potential are also associated with activation ofmacrophages. However, this process is poorly understood due to thedifficulty in applying patch clamp to motile cells. VIPs enable studiesof the electrophysiology of macrophages and other motile cells,including sperm cells for fertility studies. Thus the VIPs of theinvention can be used in methods to screen for drugs or agents thataffect, for example, immunity and immune diseases, as well as fertility.

The examples describe expression of VIPs in rat hippocampal neurons,mouse HL-1 cardiomyocytes, and human iPS-derived cardiomyocytes. In allcell types, single action potentials (APs) were readily observed. Wetested the effects of drugs on the AP waveform.

For example, in one embodiment, the invention provides a method whereinthe cell expressing a microbial rhodopsin is further exposed to astimulus capable of or suspected to be capable of changing membranepotential.

Stimuli that can be used include candidate agents, such as drugcandidates, small organic and inorganic molecules, larger organicmolecules and libraries of molecules and any combinations thereof. Onecan also use a combination of a known drug, such as an antibiotic with acandidate agent to screen for agents that may increase the effectivenessof the one or more of the existing drugs, such as antibiotics.

The methods of the invention are also useful for vitro toxicityscreening and drug development. For example, using the methods describedherein one can make a human cardiomyocyte from induced pluripotent cellsthat stably express a modified archaerhodopsin wherein the protonpumping activity is substantially reduced or abolished. Such cells areparticularly useful for in vitro toxicity screening in drug development.

PROPS: An Exemplary Optogenetic Voltage Sensor Derived from GPR

GPR has seven spectroscopically distinguishable states that it passesthrough in its photocycle. In principle the transition between any pairof states is sensitive to membrane potential. In one embodiment, theacid-base equilibrium of the Schiff base was chosen as thewavelength-shifting transition, hence the name of the sensor:Proteorhodopsin Optical Proton Sensor (PROPS). Characterization of theproperties of PROPS and its uses are described herein in the Examplessection. A brief discussion of PROPS is provided herein below.

The absorption spectrum of wild-type GPR is known to depend sensitivelyon the state of protonation of the Schiff base. When protonated, theabsorption maximum is at 545 nm, and when deprotonated the maximum is at412 nm. When GPR absorbs a photon, the retinal undergoes a 13-trans tocis isomerization, which causes a proton to hop from the Schiff base tonearby Asp97, leading to a shift from absorption at 545 nm to 412 nm.The PROPS design described herein seeks to recapitulate this shift inresponse to a change in membrane potential.

Two aspects of wild-type GPR can be changed for it to serve as anoptimal voltage sensor. First, the pKa of the Schiff base can be beshifted from its wild-type value of ˜12 to a value close to the ambientpH. When pKa ˜pH, the state of protonation becomes maximally sensitiveto the membrane potential. Second, the endogenous charge-pumpingcapability can be be eliminated, because optimally, a voltage probeshould not perturb the quantity under study. However, in somesituations, a wild type microbial rhodopsin can be used, such as Arch 3WT, which functions in neurons to measure membrane potential as shown inour examples.

In one embodiment, a single point mutation induces both changes in GPR.Mutating Asp97 to Asn eliminates a negative charge near the Schiff base,and destabilizes the proton on the Schiff base. The pKa shifts from ˜12to 9.8. In wild-type GPR, Asp97 also serves as the proton acceptor inthe first step of the photocycle, so removing this amino acid eliminatesproton pumping. This mutant of GPR is referred to herein as PROPS.

Similarly, in an analogous voltage sensor derived from BPR, thehomologous mutation Asp99 to Asn lowers the pKa of the Schiff base andeliminates the proton-pumping photocycle. Thus, in one embodiment theoptical sensor is derived from BPR in which the amino acid residue Asp99is mutated to Asn.

In GPR, additional mutations shift the pKa closer to the physiologicalvalue of 7.4. In particular, mutations Glu108 to Gln and Glu142 to Glnindividually or in combination lead to decreases in the pKa and tofurther increases in the sensitivity to voltage. Many mutations otherthan those discussed herein may lead to additional changes in the pKaand improvements in the optical properties of PROPS and are contemplatedherein.

Expression Vectors and Targeting Sequences

Optical sensors can be expressed in a cell using an expression vector.The term “vector” refers to a carrier DNA molecule into which a nucleicacid sequence can be inserted for introduction into a host cell. An“expression vector” is a specialized vector that contains the necessaryregulatory regions needed for expression of a gene of interest in a hostcell. In some embodiments the gene of interest is operably linked toanother sequence in the vector. In some embodiments, it is preferredthat the viral vectors are replication defective, which can be achievedfor example by removing all viral nucleic acids that encode forreplication. A replication defective viral vector will still retain itsinfective properties and enters the cells in a similar manner as areplicating vector, however once admitted to the cell a replicationdefective viral vector does not reproduce or multiply. The term“operably linked” means that the regulatory sequences necessary forexpression of the coding sequence are placed in the DNA molecule in theappropriate positions relative to the coding sequence so as to effectexpression of the coding sequence. This same definition is sometimesapplied to the arrangement of coding sequences and transcription controlelements (e.g. promoters, enhancers, and termination elements) in anexpression vector.

Many viral vectors or virus-associated vectors are known in the art.Such vectors can be used as carriers of a nucleic acid construct intothe cell. Constructs may be integrated and packaged intonon-replicating, defective viral genomes like Adenovirus,Adeno-associated virus (AAV), or Herpes simplex virus (HSV) or others,including retroviral and lentiviral vectors, for infection ortransduction into cells. The vector may or may not be incorporated intothe cell's genome. The constructs may include viral sequences fortransfection, if desired. Alternatively, the construct may beincorporated into vectors capable of episomal replication, e.g EPV andEBV vectors. The inserted material of the vectors described herein maybe operatively linked to an expression control sequence when theexpression control sequence controls and regulates the transcription andtranslation of that polynucleotide sequence. The term “operativelylinked” includes having an appropriate start signal (e.g., ATG) in frontof the polynucleotide sequence to be expressed, and maintaining thecorrect reading frame to permit expression of the polynucleotidesequence under the control of the expression control sequence, andproduction of the desired polypeptide encoded by the polynucleotidesequence. In some examples, transcription of an inserted material isunder the control of a promoter sequence (or other transcriptionalregulatory sequence) which controls the expression of the recombinantgene in a cell-type in which expression is intended. It will also beunderstood that the inserted material can be under the control oftranscriptional regulatory sequences which are the same or which aredifferent from those sequences which control transcription of thenaturally-occurring form of a protein. In some instances the promotersequence is recognized by the synthetic machinery of the cell, orintroduced synthetic machinery, required for initiating transcription ofa specific gene.

An “inducible promoter” is a promoter that is capable of directly orindirectly activating transcription of one or more DNA sequences orgenes in response to a “regulatory agent” (e.g., doxycycline), or a“stimulus” (e.g., heat). In the absence of a “regulatory agent” or“stimulus”, the DNA sequences or genes will not be substantiallytranscribed. The term “not substantially transcribed” or “notsubstantially expressed” means that the level of transcription is atleast 100-fold lower than the level of transcription observed in thepresence of an appropriate stimulus or regulatory agent; preferably atleast 200-fold, 300-fold, 400-fold, 500-fold or more. As used herein,the terms “stimulus” and/or “regulatory agent” refers to a chemicalagent, such as a metabolite, a small molecule, or a physiological stressdirectly imposed upon the organism such as cold, heat, toxins, orthrough the action of a pathogen or disease agent. A recombinant cellcontaining an inducible promoter may be exposed to a regulatory agent orstimulus by externally applying the agent or stimulus to the cell ororganism by exposure to the appropriate environmental condition or theoperative pathogen. Inducible promoters initiate transcription only inthe presence of a regulatory agent or stimulus. Examples of induciblepromoters include the tetracycline response element and promotersderived from the β-interferon gene, heat shock gene, metallothioneingene or any obtainable from steroid hormone-responsive genes. Induciblepromoters which may be used in performing the methods of the presentinvention include those regulated by hormones and hormone analogs suchas progesterone, ecdysone and glucocorticoids as well as promoters whichare regulated by tetracycline, heat shock, heavy metal ions, interferon,and lactose operon activating compounds. For review of these systems seeGingrich and Roder, 1998, Annu Rev Neurosci 21, 377-405. Tissue specificexpression has been well characterized in the field of gene expressionand tissue specific and inducible promoters are well known in the art.These promoters are used to regulate the expression of the foreign geneafter it has been introduced into the target cell.

The promoter sequence may be a “cell-type specific promoter” or a“tissue-specific promoter” which means a nucleic acid sequence thatserves as a promoter, i.e., regulates expression of a selected nucleicacid sequence operably linked to the promoter, and which affectsexpression of the selected nucleic acid sequence in specific cells ortissues where membrane potential is desired to be measured. In someembodiments, the cell-type specific promoter is a leaky cell-typespecific promoter. The term “leaky” promoter refers to a promoter whichregulates expression of a selected nucleic acid primarily in one celltype, but cause expression in other cells as well. For expression of anexogenous gene specifically in neuronal cells, a neuron-specific enolasepromoter can be used (see Forss-Petter et al., 1990, Neuron 5: 187-197).For expression of an exogenous gene in dopaminergic neurons, a tyrosinehydroxylase promoter can be used. For expression in pituitary cells, apituitary-specific promoter such as POMC may be used (Hammer et al.,1990, Mol. Endocrinol. 4:1689-97). Examples of muscle specific promotersinclude, for example α-myosin heavy chain promoter, and the MCKpromoter. Other cell specific promoters active in mammalian cells arealso contemplated herein. Such promoters provide a convenient means forcontrolling expression of the exogenous gene in a cell of a cell cultureor within a mammal.

In some embodiments, the expression vector is a lentiviral vector.Lentiviral vectors useful for the methods and compositions describedherein can comprise a eukaryotic promoter. The promoter can be anyinducible promoter, including synthetic promoters, that can function asa promoter in a eukaryotic cell. For example, the eukaryotic promotercan be, but is not limited to, ecdysone inducible promoters, E1ainducible promoters, tetracycline inducible promoters etc., as are wellknown in the art. In addition, the lentiviral vectors used herein canfurther comprise a selectable marker, which can comprise a promoter anda coding sequence for a selectable trait. Nucleotide sequences encodingselectable markers are well known in the art, and include those thatencode gene products conferring resistance to antibiotics oranti-metabolites, or that supply an auxotrophic requirement. Examples ofsuch sequences include, but are not limited to, those that encodethymidine kinase activity, or resistance to methotrexate, ampicillin,kanamycin, chloramphenicol, puromycinor zeocin, among many others.

In some embodiments the viral vector is an adeno-associated virus (AAV)vector. AAV can infect both dividing and non-dividing cells and mayincorporate its genome into that of the host cell.

The type of vector one selects will also depend on whether theexpression is intended to be stable or transient.

The invention also provides cells that are genetically engineered toexpress the microbial rhodopsin, such as VIPs or PROPS. The cell may beengineered to express the VIP or PROPS transiently or stably.

The invention provides methods of making both transiently expressingcells and cells and cell lines that express the microbial rhodopsinsstably.

Transient expression. One of ordinary skill in the art is well equippedto engineer cells that are transiently transfected to express the VIPsor PROPS as described herein. Transduction and transformation methodsfor transient expression of nucleic acids are well known to one skilledin the art.

Transient transfection can be carried out, e.g., using calciumphosphate, by electroporation, or by mixing a cationic lipid with thematerial to produce liposomes, cationic polymers or highly branchedorganic compounds. All these are in routine use in genetic engineering.

Stable expression of VIP or PROP in an eukaryotic cell. One of ordinaryskill in the art is well equipped to engineer cells that stably expressthe VIPs or PROPS as described herein. These methods are also in routineuse in genetic engineering. Exemplary protocols can be found, e.g., inEssential Stem Cell Methods, edited by Lanza and Klimanskaya, publishedin 2008, Academic Press. For example, one can generate a virus thatintegrates into the genome and comprises a selectable marker, and infectthe cells with the virus and screen for cells that express the marker,which cells are the ones that have incorporated the virus into theirgenome. For example, one can generate a VSV-g psuedotyped lenti viruswith a puromycin selectable marker in HEK cells according to establishedprocedures. Generally, one can use a stem cell specific promoter toencode a GFP if FACS sorting is necessary. The hiPS cultures arecultivated on embryonic fibroblast (EF) feeder layers or on Matrigel infibroblast growth factor supplemented EF conditioned medium. The cellsare dissociated by trypsinization to a single cell suspension The cellscan be plated, e.g., 1×10⁵ cells on a tissue culture 6-well platepretreated with, e.g., Matrigel. To maintain the cells in anundifferentiated state, one can use, e.g., EF conditioned medium. About6 hours after plating, one can add virus supernatant to adhered cells(use 5×10⁶ IU virus per 1×10⁵ cells). Add 6 μg/mL protamine sulfate toenhance virus infection. Cells are cultured with the virus for 24 hours;washed, typically with PBS, and fresh media is added with a selectionmarker, such as 1 μg/mL puromycin. The medium is replaced about every 2days with additional puromycin. Cells surviving after 1 week arere-plated, e.g., using the hanging drop method to form EBs with stableincorporation of gene.

In some embodiments, it is advantageous to express an optical voltagesensor (e.g., Arch 3 D94N) in only a single cell-type within anorganism, and further, if desired, to direct the sensor to a particularsubcellular structure within the cell. Upstream promoters control whenand where the gene is expressed. Constructs are made that optimizeexpression in all eukaryotic cells. In one embodiment, the opticalvoltage sensor is under the control of a neuron-specific promoter.

The promoter sequence can be selected to restrict expression of theprotein to a specific class of cells and environmental conditions.Common promoter sequences include, but are not limited to, CMV(cytomegalovirus promoter; a universal promoter for mammalian cells),14× UAS-E1b (in combination with the transactivator Gal4, this promoterallows combinatorial control of transgene expression in a wide array ofeukaryotes. Tissue-specific expression can be achieved by placing Gal4under an appropriate promoter, and then using Gal4 to drive theUAS-controlled transgene), HuC (drives pan-neuronal expression inzebrafish and other teleosts), ara (allows regulation of expression witharabinose in bacteria) and lac (allows regulation of expression withIPTG in bacteria).

In some embodiments, the optical voltage sensor further comprises alocalization or targeting sequence to direct or sort the sensor to aparticular face of a biological membrane or subcellular organelle.Preferred localization sequences provide for highly specificlocalization of the protein, with minimal accumulation in othersubcellular compartments. Example localization sequences that directproteins to specific subcellular structures are shown below in Table 4.

TABLE 4 Exemplary protein localization sequences Subcellular compartmentSequence SEQ ID NO. Nuclear (import signal) PPKKKRKV  1Endoplasmic reticulum MSFVSLLLVGILFWATGAENLTKCEVFN  2 (import signal)Endoplasmic reticulum KDEL  3 (retention signal) Peroxisome (import SKL 4 signal) Peroxisome (import (R/K)-(L/V/I)-XXXXX-(H/Q)-(where  5signal) (L/A/F) X can be any amino acid). Mitochondrial innerMLSLRNSIRFFKPATRTLCSSRYLL  6 membrane Mitochondrial outerMLRTSSLFTRRVQPSLFRNILRLQST  7 membrane plasma membraneMGCIKSKRKDNLNDDGVDMKT  8 (cytosolic face) plasma membraneKKKKKKKSKTKCVIM  9 (cytosolic face) mitochondrial targetingMAVRQALGRGLQLGRALLLR 10 sequence: human FTGKPGRAYGLGRPGPAAGC PINK1VRGERPGWAAGPGAEPRRVG LGLPNRLRFFRQSVAGL mitochondrial targetingMAAPRAGRGAGWSLRAWRAL 11 sequence: human serine GGIRWGRRPRLprotease HTRA2 mitochondrial targeting MFADRWLFST NHKDIGTLY 12sequence: human cytochrome oxidase 1 mitochondrial targetingMAHAAQVGLQ DATSPIMEEL ITFHDH 13 sequence: human cytochrome oxidase 2mitochondrial targeting MSTAALITL VRSGGNQVRRR 14 sequence: humanVLLSSRLLQ protein phospatase 1K mitochondrial targetingMLSVRVAAAVVRALPRRAGL 15 sequence: human ATP VSRNALGSSFIAARNFHASNTHLsynthase alpha mitochondrial targeting MWTLGRRAVAGLLASPSPAQ 16sequence: human AQTLTRVPRPAELAPLCGRRG frataxin

Other examples of localization signals are described in, e.g., “ProteinTargeting”, chapter 35 of Stryer, L., Biochemistry (4th ed.). W. H.Freeman, 1995 and Chapter 12 (pages 551-598) of Molecular Biology of theCell, Alberts et al. third edition, (1994) Garland Publishing Inc. Insome embodiments, more than one discrete localization motif is used toprovide for correct sorting by the cellular machinery. For example,correct sorting of proteins to the extracellular face of the plasmamembrane can be achieved using an N-terminal signal sequence and aC-terminal GPI anchor or transmembrane domain.

Typically, localization sequences can be located almost anywhere in theamino acid sequence of the protein. In some cases the localizationsequence can be split into two blocks separated from each other by avariable number of amino acids. The creation of such constructs viastandard recombinant DNA approaches is well known in the art, as forexample described in Maniatis, et al., Molecular Cloning A LaboratoryManual, Cold Spring Harbor Laboratory, N.Y, 1989).

Targeting to the plasma membrane: In some embodiments, constructs aredesigned to include signaling sequences to optimize localization of theprotein to the plasma membrane. These can include e.g., a C-terminalsignaling sequence from the □2 nicotinic acetylcholine receptor(MRGTPLLLVVSLFSLLQD (SEQ ID NO: 17), indicated with the one-letter aminoacid code), and/or an endoplasmic reticulum export motif from Kir2.1,(FCYENEV) (SEQ ID NO: 18).

Additional improvements in plasma localization can be obtained by addingGolgi export sequences (e.g. from Kir2.1: RSRFVKKDGHCNVQFINV (SEQ ID NO:19)) and membrane localization sequences (e.g. from Kir2.1:KSRITSEGEYIPLDQIDINV (SEQ ID NO: 20)) (Gradinaru, V. et al. Cell(2010)). In some embodiments, the targeting sequence is selected toregulate intracellular transport of the protein to the desiredsubcellular structure. In one embodiment the protein is targeted to theplasma membrane of a eukaryotic cell. In this case the targetingsequence can be designed following the strategy outlined in e.g.,Gradinaru et al., “Molecular and Cellular Approaches for Diversifyingand Extending Optogenetics,” Cell 141, 154-165 (2010). The term “signalsequence” refers to N-terminal domains that target proteins into asubcellular locale e.g., the endoplasmic reticulum (ER), and thus are ontheir way to the plasma membrane. Signal sequences used in optogeneticvoltage sensors can be derived from the proteins β2-n-acetylcholinereceptor (SS B2nAChR) and PPL. In addition, there is an endogenoussignaling sequence on microbial rhodopsin proteins that can be harnessedfor appropriate subcellular targeting. A trafficking signal (TS) canoptionally be inserted into the genome C-terminal to the microbialrhodopsin and N-terminal to the accessory fluorescent protein. In oneembodiment, the trafficking signal is derived from the Kir2.1 protein asspecified in Gradinaru et al. In another embodiment, an ER export motifis inserted at the C-terminus of the accessory fluorescent protein.

Targeting mitochondria: For measuring mitochondrial membrane potentialor for studying mitochondria, one may wish to localize PROPS to themitochondrial inner membrane or mitochondrial outer membrane, in whichcase appropriate signaling sequences can be added to the rhodopsinprotein.

Optogenetic voltage sensors can be targeted to the inner mitochondrialmembrane, following a procedure such as that described in e.g., A.Hoffmann, V. Hildebrandt, J. Heberle, and G. MIldt, “Photoactivemitochondria: in vivo transfer of a light-driven proton pump into theinner mitochondrial membrane of Schizosaccharomyces pombe,” Proc. Natl.Acad. Sci. USA 91, PNAS 9367-9371 (1994).

Cells

Cells that are useful according to the invention include eukaryotic andprokaryotic cells. Eukaryotic cells include cells of non-mammalianinvertebrates, such as yeast, plants, and nematodes, as well asnon-mammalian vertebrates, such as fish and birds. The cells alsoinclude mammalian cells, including human cells. The cells also includeimmortalized cell lines such as HEK, HeLa, CHO, 3T3, which may beparticularly useful in applications of the methods for drug screens. Thecells also include stem cells, pluripotent cells, progenotir cells, andinduced pluripotent cells. Differentiated cells including cellsdifferentiated from the stem cells, pluripotent cells and progenotircells are included as well.

In some embodiments, the cells are cultured in vitro or ex vivo. In someembodiments, the cells are part of an organ or an organism.

In some embodiment, the cell is an “artificial cell” or a “syntheticcell” created by bioengineering (see, e.g., Creation of a Bacterial CellControlled by a Chemically Synthesized Genome, Daniel G. Gibson et al.,Science 2 Jul. 2010: Vol. 329 no. 5987 pp. 52-56; Cans, Ann-Sofie,Andes-Koback, Meghan, and Keating, Christine D. Positioning LipidMembrane Domains in Giant Vesicles by Micro-organization of AqueousCytoplasm Mimic. J. Am. Chem. Soc., 2008).

The methods can also be applied to any other membrane-bound structure,which may not necessarily be classified as a cell. Such membrane boundstructures can be made to carry the microbial rhodopsin proteins of theinvention by, e.g., fusing the membranes with cell membrane fragmentsthat carry the microbial rhodopsin proteins of the invention.

Cells include also zebrafish cardiomyocytes; immune cells (primarymurine and human cultures and iPS-derived lines for all, in addition tothe specific lines noted below), including B cells (e.g., human Rajicell line, and the DT40 chicken cell line), T cells (e.g., human Jurkatcell line), Macrophages, Dendritic cells, and Neutrophils (e.g., HL-60line). Additionally, one can use glial cells: astrocytes andoligodendrocytes; pancreatic beta cells; hepatocytes; non-cardiac musclecells; endocrine cells such as parafollicular and chromaffin; and yeastcells. Cells further include neuronal cells, such as neurons.

The cell can also be a Gram positive or a Gram negative bacteria, aswell as pathogenic bacteria of either Gram type. The pathogenic cellsare useful for applications of the method to, e.g., screening of novelantibiotics that affect membrane potential to assist in destruction ofthe bacterial cell or that affect membrane potential to assistdestruction of the bacterial cell in combination with the membranepotential affecting agent; or in the search for compounds that suppressefflux of antibiotics.

The membrane potential of essentially any cell, or any phospholipidbilayer enclosed structure, can be measured using the methods andcompositions described herein. Examples of the cells that can be assayedare a primary cell e.g., a primary hepatocyte, a primary neuronal cell,a primary myoblast, a primary mesenchymal stem cell, primary progenitorcell, or it may be a cell of an established cell line. It is notnecessary that the cell be capable of undergoing cell division; aterminally differentiated cell can be used in the methods describedherein. In this context, the cell can be of any cell type including, butnot limited to, epithelial, endothelial, neuronal, adipose, cardiac,skeletal muscle, fibroblast, immune cells, hepatic, splenic, lung,circulating blood cells, reproductive cells, gastrointestinal, renal,bone marrow, and pancreatic cells. The cell can be a cell line, a stemcell, or a primary cell isolated from any tissue including, but notlimited to brain, liver, lung, gut, stomach, fat, muscle, testes,uterus, ovary, skin, spleen, endocrine organ and bone, etc. Where thecell is maintained under in vitro conditions, conventional tissueculture conditions and methods can be used, and are known to those ofskill in the art. Isolation and culture methods for various cells arewell within the knowledge of one skilled in the art. The cell can be aprokaryotic cell, a eukaryotic cell, a mammalian cell or a human cell.In one embodiment, the cell is a neuron or other cell of the brain. Insome embodiment, the cell is a cardiomyocyte. In some embodiments, thecell is cardiomyocyte that has been differentiated from an inducedpluripotent cell.

Reference Value

The invention provides method for measuring membrane potential in a cellexpressing a nucleic acid encoding a microbial rhodopsin protein, themethod comprising the steps of (a) exciting at least one cell comprisinga nucleic acid encoding a microbial rhodopsin protein with light of atleast one wave length; and (b) detecting at least one optical signalfrom the at least one cell, wherein the level of fluorescence emitted bythe at least one cell compared to a reference is indicative of themembrane potential of the cell.

The term “reference” as used herein refers to a baseline value of anykind that one skilled in the art can use in the methods. In someembodiments, the reference is a cell that has not been exposed to astimulus capable of or suspected to be capable of changing membranepotential. In one embodiment, the reference is the same cell transfectedwith the microbial rhodopsin but observed at a different time point. Inanother embodiment, the reference is the fluorescence of a homologue ofGreen Fluorescent Protein (GFP) operably fused to the microbialrhodopsin.

Detecting Fluorescence from a Modified Microbial Rhodopsin

In the methods of the invention, the cells are excited with a lightsource so that the emitted fluorescence can be detected. The wavelengthof the excitation light depends on the fluorescent molecule. Forexample, the archerhodopsin constructs in the examples are all excitableusing light with wavelengths varying between λ=594 nm and λ=645 nm.Alternatively, the range may be between λ=630-645 nm. For example acommonly used Helium Neon laser emits at λ=632.8 nm and can be used inexcitation of the fluorescent emission of these molecules.

In some embodiments a second light is used. For example, if the cellexpresses a reference fluorescent molecule or a fluorescent moleculethat is used to detect another feature of the cell, such a pH or Calciumconcentration. In such case, the second wavelength differs from thefirst wavelength. Examples of useful wavelengths include wavelengths inthe range of λ=447-594 nm, for example, λ=473 nm, λ=488 nm, λ=514 nm,λ=532 nm, and λ=561 nm.

The hardware and software needed to take maximal advantage of VIPsdepends on the type of assay, and can be easily optimized and selectedby a skilled artisan based on the information provided herein. Existinginstrumentation can be easily used or adapted for the detection of VIPsand PROPs. The factors that determine the type of instrumentationinclude, precision and accuracy, speed, depth penetration, multiplexingand throughput.

Precision and accuracy: In determining the detection system, one shouldevaluate whether one needs absolute or relative measurement of voltage.Absolute measurements of membrane potential are typically doneratiometrically, with either two excitation wavelengths or two emissionwavelengths. Relative voltage changes in a single cell can be performedwith single-band excitation and detection.

Speed: To measure action potentials in a neuron one needssub-millisecond temporal resolution. This requires either high-speedCCDs, or high-speed confocal microscopes which can scan customtrajectories. Slower dynamics and quasi steady state voltages can bemeasured with conventional cameras. These measurements can be used, forexample, in methods and assays that are directed to screening of agentsin cardiac cells, such as cardiomyocytes.

Depth penetration: Imaging in deep tissue may require confocalmicroscopy or lateral sheet illumination microscopy. Alternatively, deepimaging may require the development of nonlinear microscopies, includingtwo-photon fluorescence or second harmonic generation. Conventionalepifluorescence imaging works well for cells in culture, and totalinternal reflection fluorescence (TIRF) provides particularly highsignal-to-noise ratios in images of adherent cells.

Multiplexing with other optical imaging and control: One can combineimaging of VIPs with other structural and functional imaging, of e.g.pH, calcium, or ATP. One may also combine imaging of VIPs withoptogenetic control of membrane potential using e.g. channelrhodopsin,halorhodopsin, and archaerhodopsin. If optical measurement and controlare combined in a feedback loop, one can perform all-optical patch clampto probe the dynamic electrical response of any membrane.

Throughput: One can also integrate robotics and custom software forscreening large libraries or large numbers of conditions which aretypically encountered in high throughput drug screening methods.

Spectroscopic Readouts of Voltage-Induced Shifts in Microbial Rhodopsins

The spectroscopic states of microbial rhodopsins are typicallyclassified by their absorption spectrum. However, in some cases there isinsufficient protein in a single cell to detect spectral shifts viaabsorbance alone. Any of the following several optical imagingtechniques can be used to probe other state-dependent spectroscopicproperties.

a) Fluorescence

It was found that many microbial rhodopsin proteins and their mutantsproduce measurable fluorescence. For example, PROPS fluorescence isexcited by light with a wavelength between wavelength of 500 and 650 nm,and emission is peaked at 710 nm. The rate of photobleaching of PROPSdecreases at longer excitation wavelengths, so one preferable excitationwavelength is in the red portion of the spectrum, near 633 nm. Thesewavelengths are further to the red than the excitation and emissionwavelengths of any other fluorescent protein, a highly desirableproperty for in vivo imaging. Furthermore, the fluorescence of PROPSshows negligible photobleaching, in stark contrast to all other knownfluorophores. When excited at 633 nm, PROPS and GFP emit a comparablenumbers of photons prior to photobleaching. Thus microbial rhodopsinsconstitute a new class of highly photostable, membrane-bound fluorescentmarkers.

It was further found that the fluorescence of PROPS is exquisitelysensitive to the state of protonation of the Schiff base in that onlythe protonated form fluoresces. Thus voltage-induced changes inprotonation lead to changes in fluorescence.′

In some embodiments, the fluorescence of PROPS is detected using e.g., afluorescent microscope, a fluorescent plate reader, FACS sorting offluorescent cells, etc.

b) Spectral Shift Fluorescence Resonance Energy Transfer (FRET)

FRET is a useful tool to quantify molecular dynamics in biophysics andbiochemistry, such as protein-protein interactions, protein-DNAinteractions, and protein conformational changes. For monitoring thecomplex formation between two molecules (e.g., retinal and microbialrhodopsin), one of them is labeled with a donor and the other with anacceptor, and these fluorophore-labeled molecules are mixed. When theyare dissociated, the donor emission is detected upon the donorexcitation. On the other hand, when the donor and acceptor are inproximity (1-10 nm) due to the interaction of the two molecules, theacceptor emission is predominantly observed because of theintermolecular FRET from the donor to the acceptor.

A fluorescent molecule appended to a microbial rhodopsin can transferits excitation energy to the retinal, but only if the absorptionspectrum of the retinal overlaps with the emission spectrum of thefluorophore. Changes in the absorption spectrum of the retinal lead tochanges in the fluorescence brightness of the fluorophore. To performspectral shift FRET, a fluorescent protein is fused with the microbialrhodopsin voltage sensor, and the fluorescence of the protein ismonitored. This approach has the advantage over direct fluorescence thatthe emission of fluorescent proteins is far brighter than that ofretinal, but the disadvantage of being an indirect readout, with smallerfractional changes in fluorescence.

In some embodiments, voltage-induced changes in the absorption spectrumof microbial rhodopsins are detected using spectral shift FRET

c) Rhodopsin Optical Lock-in Imaging (ROLI)

The absorption spectrum of many of the states of retinal is temporarilychanged by a brief pulse of light. In ROLI, periodic pulses of a “pump”beam are delivered to the sample. A second “probe” beam measures theabsorbance of the sample at a wavelength at which the pump beam inducesa large change in absorbance. Thus the pump beam imprints a periodicmodulation on the transmitted intensity of the probe beam. Theseperiodic intensity changes are detected by a lock-in imaging system. Incontrast to conventional absorption imaging, ROLI providesretinal-specific contrast. Modulation of the pump at a high frequencyallows detection of very small changes in absorbance.

In some embodiments, the fluorescence of PROPS is detected usingrhodopsin optical lock-in imaging.

d) Raman

Raman spectroscopy is a technique that can detect vibrational,rotational, and other low-frequency modes in a system. The techniquerelies on inelastic scattering of monochromatic light (e.g., a visiblelaser, a near infrared laser or a near ultraviolet laser). Themonochromatic light interacts with molecular vibrations, phonons orother excitations in the system, resulting in an energy shift of thelaser photons. The shift in energy provides information about the phononmodes in the system.

Retinal in microbial rhodopsin molecules is known to have a strongresonant Raman signal. This signal is dependent on the electrostaticenvironment around the chromophore, and therefore is sensitive tovoltage.

In some embodiments, voltage-induced changes in the Raman spectrum ofmicrobial rhodopsins are detected using Raman microscopy.

e) Second Harmonic Generation (SHG)

Second harmonic generation, also known in the art as “frequencydoubling” is a nonlinear optical process, in which photons interactingwith a nonlinear material are effectively “combined” to form new photonswith twice the energy, and therefore twice the frequency and half thewavelength of the initial photons.

SHG signals have been observed from oriented films of bacteriorhodopsinin cell membranes. SHG is an effective probe of the electrostaticenvironment around the retinal in optical voltage sensors. Furthermore,SHG imaging involves excitation with infrared light which penetratesdeep into tissue. Thus SHG imaging can be used for three-dimensionaloptical voltage sensing using the optical sensors described herein.

In some embodiments, voltage-induced changes in the second harmonicspectrum of microbial rhodopsins are detected using SHG imaging.

Fusion Protein with a Moiety that Produces an Optical Signal

Although microbial rhodopsin proteins are themselves fluorescent inresponse to changes in voltage, in some applications it may desired ornecessary to enhance the level of fluorescence or provide anotheroptical signal (e.g., a colorimetric signal) to permit detection ofvoltage changes. Further, a moiety that produces an optical signal canbe attached to the microbial rhodopsin to monitor the subcellularlocalization of the rhodopsin protein. Thus, in some embodiments, themodified microbial rhodopsin proteins further comprise a moiety thatproduces an optical signal, thereby enhancing the optical signalmeasured from the modified microbial rhodopsin protein or permittinglocalization studies to be performed for the rhodopsin protein.

For example, a gene for a fluorescent protein of the GFP family or ahomolog thereof can optionally be appended or as referred to in theclaims “operably linked” to the nucleic acid encoding the microbialrhodopsin. In one embodiment, the identity of the fluorescent protein,its linker to the voltage-sensing complex, and the location of thislinker in the overall protein sequence are selected for one of twofunctions: either to serve as an indicator of the level and distributionof gene expression products; or to serve as an alternative readout ofvoltage, independent of the endogenous fluorescence of the retinal.

For example, when the fluorescent protein serves as an indicator ofprotein localization, it enables quantitative optical voltagemeasurements that are not confounded by cell-to-cell variation inexpression levels. The fluorescence of the fluorescent protein and themicrobial rhodopsin can be measured simultaneously and the ratio ofthese two signals provides a concentration-independent measure ofmembrane potential.

In one embodiment, the microbial rhodopsin protein is a PROPS fusionprotein comprising a fluorescent protein. For example, an N-terminalfusion of PROPS with the fluorescent protein Venus. This proteinprovides a stable reference indicating localization of PROPS within thecell and permitting ratiometric imaging of Venus and PROPS fluorescence.Ratiometric imaging permits quantitative measurements of membranepotential because this technique is insensitive to the total quantity ofprotein within the cell. Other fluorescent proteins may be used in lieuof Venus with similar effects. In some embodiments, the fluorescentpolypeptide is selected from the group consisting of GFP, YFP, EGFP,EYFP, EBFB, DsRed, RFP and fluorescent variants thereof.

In one embodiment, the microbial rhodopsin is an archaerhodopsin fusedwith or operably linked to an additional fluorescent protein, such as aGFP or a homolog thereof.

Chromophore

In the wild, microbial rhodopsins contain a bound molecule of retinalwhich serves as the optically active element. These proteins will alsobind and fold around many other chromophores with similar structure, andpossibly preferable optical properties. Analogues of retinal with lockedrings cannot undergo trans-cis isomerization, and therefore have higherfluorescence quantum yields (Brack, T. et al. Biophys. J. 65, 964-972(1993)). Analogues of retinal with electron-withdrawing substituentshave a Schiff base with a lower pKa than natural retinal and thereforemay be more sensitive to voltage (Sheves, M., et al. Proc. Nat. Acad.Sci. U.S.A 83, 3262-3266 (1986); Rousso, I., et al. Biochemistry 34,12059-12065 (1995)). Covalent modifications to the retinal molecule maylead to optical voltage sensors with significantly improved opticalproperties and sensitivity to voltage.

Advantages of the Methods and Compositions Described Herein

The key figures of merit for an optical voltage sensor are its responsespeed and its sensitivity (fractional change in fluorescence per 100 mVchange in membrane potential). FIG. 10 compares these attributes forprevious protein-based fluorescent voltage indicators and thosecontemplated herein. Additional important attributes include the abilityto target the indicator to a particular cell type or sub-cellularstructure, photostability, and low phototoxicity.

Previous protein-based efforts focused on fusing one or more fluorescentproteins to transmembrane voltage sensing domains. A change in voltageinduces a conformational change in the voltage sensing domain, whichmoves the fluorescent proteins, and changes their fluorescence. Thereliance on conformational motion of multiple large protein domainsmakes these approaches unavoidably slow. Furthermore, the conformationalshifts of most voltage sensing domains are small, leading to smallchanges in fluorescence.

The most sensitive indicators from the VSFP 2.x family have a change influorescence of ΔF/F=10% per 100 mV. VSFP 2.x proteins respond inapproximately 100 milliseconds, far too slow to detect a 1 ms actionpotential in a neuron (Perron, A. et al. Front Mol Neurosci. 2 (2009);Mutoh, H. et al. PLoS One 4, e4555 (2009)). The SPARC family of voltagesensors has a 1 ms response time, but shows a fluorescence change of <1%per 100 mV (Baker, B. J. et al. J. Neurosci. Methods 161, 32-38 (2007);Ataka, K. & Pieribone, V. A. Biophys. J. 82, 509-516 (2002)). Prior tothe present study described herein, two decades of research onfluorescent voltage sensors had not yet yielded a protein that couldsignal individual neuronal action potentials in vivo.

Scientists have also developed small organic dyes that showvoltage-sensitive fluorescence. These lipophilic molecules incorporateinto the cell membrane where voltage leads to shifts in conformation orelectronic energy levels and thereby to changes in optical properties.These molecules respond quickly (less than 1 ms, typically), and havesensitivities as large as 34% per 100 mV, but cannot be targeted, areoften difficult to deliver, and are highly toxic (Krauthamer, V., et al.J. Fluoresc. 1, 207-213 (1991); Fromherz, P., et al. Eur. Biophys. J.37, 509-514 (2008); Sjulson, L. & Miesenbock, G. J. Neurosci. 28, 5582(2008)), see e.g. U.S. Pat. Nos. 7,867,282, 6,107,066, and 5,661,035).None of these optical voltage sensors employs a microbial rhodopsinprotein that is configured to run “backwards” to convert changes inmembrane potential into changes in an optically detectable signal.

The approach to optical voltage sensing described herein is differentfrom previous efforts. As described herein a protein is used that has astrong electro-optical coupling in the wild. Microbial rhodopsins in thewild serve to transduce sunlight into a membrane potential. The opticalvoltage sensors described herein use this function in reverse,transducing a membrane potential into a readily detectable opticalsignal. As FIG. 12 shows, the exemplary microbial rhodopsin voltagesensors (PROPS, Arch 3 WT, Arch 3 D95N) exceed other protein-basedindicators on the key figures of merit.

Membrane Fusion Mediated Delivery of an Optical Sensor

Membrane fusion reactions are common in eukaryotic cells. Membranes arefused intracellularly in processes including endocytosis, organelleformation, inter-organelle traffic, and constitutive and regulatedexocytosis. Intercellularly, membrane fusion occurs during sperm-eggfusion and myoblast fusion.

Membrane fusion has been induced artificially by the use of liposomes,in which the cell membrane is fused with the liposomal membrane, and byvarious chemicals or lipids, which induce cell-cell fusion to produceheterokaryons. Naturally occurring proteins shown to induce fusion ofbiological membranes are mainly fusion proteins of enveloped viruses.Thus, in some embodiments, the optical sensor is administered using aliposome comprising a fusogenic protein.

It is generally believed that membrane fusion under physiologicalconditions is protein-mediated. This has led to the development ofliposomes that contain fusion-promoting proteins (proteoliposomes), withdecreased cytotoxicity (see, for example, Cheng, Hum. Gene Ther.7:275-282 (1996); Hara et al., Gene 159:167-174 (1995); and Findeis etal., Trends Biotechnol., 11:202-205 (1993)).

The only proteins conclusively shown to induce intercellular fusion ofbiological membranes are those of enveloped viruses and two proteinsfrom nonenveloped viruses. All enveloped viruses encode proteinsresponsible for fusion of the viral envelope with the cell membrane.These viral fusion proteins are essential for infection of susceptiblecells. The mechanism of action of fusion proteins from enveloped viruseshave served as a paradigm for protein-mediated membrane fusion (see, forexample, White, Ann. Rev. Physiol., 52:675-697 (1990); and White,Science, 258:917-924 (1992)).

Most enveloped virus fusion proteins are relatively large, multimeric,type I membrane proteins, as typified by the influenza virus HA protein,a low pH-activated fusion protein, and the Sendai virus F protein, whichfunctions at neutral pH. These are structural proteins of the virus withthe majority of the fusion protein oriented on the external surface ofthe virion to facilitate interactions between the virus particle and thecell membrane.

According to the mechanism of action of fusion proteins from envelopedviruses, fusion of the viral envelope with the cell membrane is mediatedby an amphipathic alpha-helical region, referred to as a fusion peptidemotif, that is present in the viral fusion protein. This type of fusionpeptide motif is typically 17 to 28 residues long, hydrophobic (averagehydrophobicity of about 0.6±0.1), and contains a high content of glycineand alanine, typically 36%±7% (White, Annu. Rev. Physiol., 52:675-697(1990).

All of the enveloped virus fusion proteins are believed to function viaextensive conformational changes that, by supplying the energy toovercome the thermodynamic barrier, promote membrane fusion. Theseconformational changes are frequently mediated by heptad repeat regionsthat form coiled coil structures (see Skehel and Wiley, Cell, 95:871-874(1998)). Recognition of the importance of fusion peptide motifs intriggering membrane fusion has resulted in the use of small peptidescontaining fusion peptide motifs to enhance liposome-cell fusion (see,for example, Muga et al., Biochemistry 33:4444-4448 (1994)).

Enveloped virus fusion proteins also trigger cell-cell fusion, resultingin the formation of polykaryons (syncytia). Synthesis of the viralfusion protein inside the infected cell results in transport of thefusion protein through the endoplasmic reticulum and Golgi transportsystem to the cell membrane, an essential step in the assembly andbudding of infectious progeny virus particles from the infected cell(Petterson, Curr. Top. Micro. Immunol., 170:67-106 (1991)). Thesynthesis, transport, and folding of the fusion protein is facilitatedby a variety of components, including signal peptides to target theprotein to the intracellular transport pathway, glycosylation signalsfor N-linked carbohydrate addition to the protein, and a transmembranedomain to anchor the protein in the cell membrane. These proteins havebeen used in reconstituted proteoliposomes (‘virosomes’) for enhanced,protein-mediated liposome-cell fusion in both cell culture and in vivo(see, for example, Ramani et al., FEBS Lett., 404:164-168 (1997);Scheule et al., Am. J. Respir. Cell Mol. Biol., 13:330-343 (1995); andGrimaldi, Res. Virol., 146:289-293 (1995)).

Thus, in some embodiments of the methods and compositions describedherein, a micelle, liposome or other artificial membrane comprising themodified microbial rhodopsin and a fusion protein is administered to acell or a subject to mediate delivery of the optical sensor protein tothe cell by membrane fusion. In a preferred embodiment, the compositionfurther comprises a targeting sequence to target the delivery system toa particular cell-type. If desired, the exogenous lipid of an artificialmembrane composition can further comprise a targeting moiety (e.g.,ligand) that binds to mammalian cells to facilitate entry. For example,the composition can include as a ligand an asialoglycoprotein that bindsto mammalian lectins (e.g., the hepatic asialoglycoprotein receptor),facilitating entry into mammalian cells. Single chain antibodies, whichcan target particular cell surface markers, are also contemplated hereinfor use as targeting moieties. Targeting moieties can include, forexample, a drug, a receptor, an antibody, an antibody fragment, anaptamer, a peptide, a vitamin, a carbohydrate, a protein, an adhesionmolecule, a glycoprotein, a sugar residue or a glycosaminoglycan, atherapeutic agent, a drug, or a combination of these. A skilled artisancan readily design various targeting moieties for modifying anartificial membrane based on the intended target cell to be assessedusing an optical sensor as described herein.

For methods using membrane fusion mediated delivery, it is contemplatedthat the optical sensor to be used is expressed and produced in aheterologous expression system. Different expression vectors comprisinga nucleic acid that encodes an optical sensor or derivative as describedherein for the expression of the optical sensor can be made for use witha variety of cell types or species. The expression vector should havethe necessary 5′ upstream and 3′ downstream regulatory elements such aspromoter sequences, ribosome recognition and binding TATA box, and 3′UTR AAUAAA transcription termination sequence for efficient genetranscription and translation in the desired cell. In some embodiments,the optical sensors are made in a heterologous protein expression systemand then purified for production of lipid-mediated delivery agents forfusion with a desired cell type. In such embodiments, the expressionvector can have additional sequences such as 6×-histidine (SEQ ID NO:21), V5, thioredoxin, glutathione-S-transferase, c-Myc, VSV-G, HSV,FLAG, maltose binding peptide, metal-binding peptide, HA and “secretion”signals (e.g., Honeybee melittin Pho, BiP), which are incorporated intothe expressed recombinant optical sensor for ease of purification. Inaddition, there can be enzyme digestion sites incorporated after thesesequences to facilitate enzymatic removal of additional sequence afterthey are not needed. These additional sequences are useful for thedetection of optical sensor expression, for protein purification byaffinity chromatography, enhanced solubility of the recombinant proteinin the host cytoplasm, for better protein expression especially forsmall peptides and/or for secreting the expressed recombinant proteinout into the culture media, into the periplasm of the prokaryotebacteria, or to the spheroplast of yeast cells. The expression ofrecombinant optical sensors can be constitutive in the host cells or itcan be induced, e.g., with copper sulfate, sugars such as galactose,methanol, methylamine, thiamine, tetracycline, infection withbaculovirus, and (isopropyl-beta-D-thiogalactopyranoside) IPTG, a stablesynthetic analog of lactose, depending on the host and vector systemchosen.

Examples of other expression vectors and host cells are the pET vectors(Novagen), pGEX vectors (Amersham Pharmacia), and pMAL vectors (NewEngland labs. Inc.) for protein expression in E. coli host cells such asBL21, BL21(DE3) and AD494(DE3) pLysS, Rosetta (DE3), and Origami(DE3)(Novagen); the strong CMV promoter-based pcDNA3.1 (Invitrogen) andpClneo vectors (Promega) for expression in mammalian cell lines such asCHO, COS, HEK-293, Jurkat, and MCF-7; replication incompetent adenoviralvector vectors pAdeno X, pAd5F35, pLP-Adeno-X-CMV (Clontech),pAd/CMV/V5-DEST, pAd-DEST vector (Invitrogen) for adenovirus-mediatedgene transfer and expression in mammalian cells; pLNCX2, pLXSN, andpLAPSN retrovirus vectors for use with the Retro-X™ system from Clontechfor retroviral-mediated gene transfer and expression in mammalian cells;pLenti4/V5-DEST™, pLenti6/V5-DEST™, and pLenti6.2/V5-GW/lacZ(Invitrogen) for lentivirus-mediated gene transfer and expression inmammalian cells; adenovirus-associated virus expression vectors such aspAAV-MCS, pAAV-IRES-hrGFP, and pAAV-RC vector (Stratagene) foradeno-associated virus-mediated gene transfer and expression inmammalian cells; BACpak6 baculovirus (Clontech) and pFastBac™ HT(Invitrogen) for the expression in Spodopera frugiperda 9 (Sf9) and Sf11insect cell lines; pMT/BiP/V5-His (Invitrogen) for the expression inDrosophila Schneider S2 cells; Pichia expression vectors pPICZα, pPICZ,pFLDα and pFLD (Invitrogen) for expression in Pichia pastoris andvectors pMETα and pMET for expression in P. methanolica; pYES2/GS andpYD1 (Invitrogen) vectors for expression in yeast Saccharomycescerevisiae. Recent advances in the large scale expression heterologousproteins in Chlamydomonas reinhardtii are described by Griesbeck C. et.al. 2006 Mol. Biotechnol. 34:213-33 and Fuhrmann M. 2004, Methods MolMed. 94:191-5. Foreign heterologous coding sequences are inserted intothe genome of the nucleus, chloroplast and mitochondria by homologousrecombination. The chloroplast expression vector p64 carrying theversatile chloroplast selectable marker aminoglycoside adenyltransferase (aadA), which confers resistance to spectinomycin orstreptomycin, can be used to express foreign protein in the chloroplast.The biolistic gene gun method can be used to introduce the vector in thealgae. Upon its entry into chloroplasts, the foreign DNA is releasedfrom the gene gun particles and integrates into the chloroplast genomethrough homologous recombination.

Cell-free expression systems are also contemplated. Cell-free expressionsystems offer several advantages over traditional cell-based expressionmethods, including the easy modification of reaction conditions to favorprotein folding, decreased sensitivity to product toxicity andsuitability for high-throughput strategies such as rapid expressionscreening or large amount protein production because of reduced reactionvolumes and process time. The cell-free expression system can useplasmid or linear DNA. Moreover, improvements in translation efficiencyhave resulted in yields that exceed a milligram of protein permilliliter of reaction mix. An example of a cell-free translation systemcapable of producing proteins in high yield is described by Spirin A S.et. al., Science 242:1162 (1988). The method uses a continuous flowdesign of the feeding buffer which contains amino acids, adenosinetriphosphate (ATP), and guanosine triphosphate (GTP) throughout thereaction mixture and a continuous removal of the translated polypeptideproduct. The system uses E. coli lysate to provide the cell-freecontinuous feeding buffer. This continuous flow system is compatiblewith both prokaryotic and eukaryotic expression vectors. As an example,large scale cell-free production of the integral membrane protein EmrEmultidrug transporter is described by Chang G. el. al., Science310:1950-3 (2005). Other commercially available cell-free expressionsystems include the Expressway™ Cell-Free Expression Systems(Invitrogen) which utilize an E. coli-based in-vitro system forefficient, coupled transcription and translation reactions to produce upto milligram quantities of active recombinant protein in a tube reactionformat; the Rapid Translation System (RTS) (Roche Applied Science) whichalso uses an E. coli-based in-vitro system; and the TNT CoupledReticulocyte Lysate Systems (Promega) which uses a rabbitreticulocyte-based in-vitro system.

Applications of Optical Voltage Sensors

Provided herein are areas in which an improved optical voltage indicatorcan be applied both in commercial and scientific endeavors.

Drug Screens

A recent article reported that “Among the 100 top-selling drugs, 15 areion-channel modulators with a total market value of more than $15billion.” (Molokanova, E. & Savchenko, A. Drug Discov. Today 13, 14-22(2008)). However, searches for new ion-channel modulators are limited bythe absence of good indicators of membrane potential (Przybylo, M., etal. J. Fluoresc., 1-19 (2010)). In some embodiments, the optical sensorsdescribed herein are used to measure or monitor membrane potentialchanges in response to a candidate ion channel modulator. Such screeningmethods can be performed in a high throughput manner by simultaneouslyscreening multiple candidate ion channel modulators in cells.

Stem Cells

Many genetically determined diseases of the nervous system and heartlack good animal models. In some embodiments, the optical sensorsdescribed herein are expressed in stem cells, either induced pluripotentor stem cells isolated from cord blood or amniotic fluid, or embryonicstem cells derived from humans or fetuses known to carry or be affectedwith a genetic defect. In some embodiments, the embryonal stem cells areof non-human origin. Alternatively the optical sensors are expressed inprogeny of the stem cells, either progenitor cells or differentiatedcell types, such as cardiac or neuronal cells. Expression of voltageindicators in these cell types provides information on theelectrophysiology of these cells and the response of membrane potentialto candidate agents or to changes in ambient conditions (e.g. anoxia).Additionally, expression of voltage indicators in stem cells enablesstudies of the differentiation and development of stem cells intoelectrically active cell types and tissues.

The genetic defect may be a single gene alteration, or a deletion,insertion, duplication or a rearrangement or one or more nucleic acids,including large chromosomal alterations.

Stem cells may be isolated and manipulated according to methods known toone skilled in the art. Patents describing methods of making and using,e.g., primate embryonic stem cells are described in, e.g., U.S. Pat.Nos. 7,582,479; 6,887,706; 6,613,568; 6,280,718; 6,200,806; and5,843,780. Additionally, for example, human cord blood derivedunrestricted somatic stem cells are described in U.S. Pat. No. 7,560,280and progenitor cells from wharton's jelly of human umbilical cord inU.S. Pat. No. 7,547,546.

Induced pluripotent stem cells may be produced by methods described, forexample, in U.S. Patent Application Publication No. 20110200568,European Patent Application Publication No. 01970446, and U.S. PatenApplication Publication No. US52008/0233610. Additional methods formaking and using induced pluripotent stem cells are also described inapplication U.S. Ser. No. 10/032,191, titled “Methods for cloningmammals using reprogrammed donor chromatin or donor cells,” and Ser. No.10/910,156, “Methods for altering cell fate.” These patent applicationsrelate to technology to alter the state of a cell, such as a human skincell, by exposing the cell's DNA to the cytoplasm of anotherreprogramming cell with differing properties. Detailed description ofthe reprogramming factors used in making induced pluripotent stem cells,including expression of genes OCT4, SOX2, NANOG, cMYC, LIN28 can also befound, for example, in PCT/US2006/030632.

Methods for differentiating stem cells or pluripotent cells intodifferentiated cells are also well known to one skilled in the art.

Brain Imaging

The human brain functions by sending electrical impulses along its ˜10¹¹neurons. These patterns of firing are the origin of every human thoughtand action. Yet there is currently no good way to observe large-scalepatterns of electrical activity in an intact brain (Baker, B. J. et al.J. Neurosci. Methods 161, 32-38 (2007); Baker, B. J. et al. Brain CellBiology 36, 53-67 (2008)).

An improved optical voltage sensor can lead to unprecedented insights inneuroscience. The device can allow mapping of brain activity in patientsand/or cells of patients with psychiatric and neurological diseases, andin victims of traumatic injuries or animal models modeling such diseasesand injuries.

Optical imaging of neuronal activity can also form the basis forimproved brain-machine interfaces for people with disabilities. Forimaging in the brain, the optical sensor is administered by directinjection into the site to be analyzed (with or without accompanyingelectroporation) or the optical sensor is delivered using a viralvector. Alternatively the optical sensor may be administered through theformation of a transgenic organism, or through application of theCre-Lox recombination system.

Microbiology

Bacteria are host to dozens of ion channels of unknown function(Martinac, B., et al. Physiol. Rev. 88, 1449 (2008)). Most bacteria aretoo small for direct electrophysiological measurements, so theirelectrical properties are almost entirely unknown.

Upon expressing PROPS in E. coli, it was found that E. coli undergo apreviously unknown electrical spiking behavior. The data describedherein in the Examples section is the first report of spontaneouselectrical spiking in any bacterium. This result establishes theusefulness of voltage sensors in microbes.

Furthermore, we found that electrical spiking in E. coli is coupled toefflux of a cationic membrane permeable dye. It is thus plausible thatelectrical spiking is correlated to efflux of other cationic compounds,including antibiotics. Optical voltage indicators may prove useful inscreens for inhibitors of antibiotic efflux.

Optical voltage sensors will unlock the electrophysiology of themillions of species of microorganisms which have proven too small toprobe via conventional electrophysiology. This information will beuseful for understanding the physiology of bacteria with medical,industrial, and ecological applications.

Mitochondria and Metabolic Diseases

Mitochondria are membrane-bound organelles which act as the ATPfactories in eukaryotic cells. A membrane voltage powers themitochondrial ATP synthase. Dysfunction of mitochondria has beenimplicated in a variety of neurodegenerative diseases, diabetes, cancer,cardiovascular disease, and aging. Thus there is tremendous interest inmeasuring mitochondrial membrane potential in vivo, although currentlyavailable techniques are severely limited (Verburg, J. & Hollenbeck, P.J. J. Neurosci. 28, 8306 (2008); Ichas, F., et al. Cell 89, 1145-1154(1997); Johnson, L. V., et al. Proc. Natl. Acad. Sci. U.S.A 77, 990(1980)).

The exemplary optical voltage sensor described herein (PROPS) can betagged with peptide sequences that direct it to the mitochondrial innermembrane (Hoffmann, A., et al. Proc. Nat. Acad. Sci. U.S.A 91, 9367(1994)) or the mitochondrial outer membrane, where it serves as anoptical indicator of mitochondrial membrane potential.

Imaging in Human Cells and Vertebrate Models (e.g., Rat, Mouse,Zebrafish)

As described in Example 2, we also expressed Arch 3 in HEK293T cells.Fluorescence of Arch 3 in HEK 293T cells was readily imaged in aninverted fluorescence microscope with red illumination (λ=640 nm, I=540W/cm2), a high numerical aperture objective, a Cy5 filter set, and anEMCCD camera. The cells exhibited fluorescence predominantly localizedto the plasma membrane (FIG. 6C). Cells not expressing Arch 3 were notfluorescent. Cells showed 17% photobleaching over a continuous 10-minuteexposure, and retained normal morphology during this interval.

The fluorescence of HEK cells expressing Arch 3 was highly sensitive tomembrane potential, as determined via whole-cell voltage clamp. Wedeveloped an algorithm to combine pixel intensities in a weighted sumsuch that the output, was a nearly optimal estimate of membranepotential V determined by conventional electrophysiology. FIG. 6C showsan example of a pixel-weight matrix, indicating that thevoltage-sensitive protein was localized to the cell membrane;intracellular Arch 3 contributed fluorescence but no voltage-dependentsignal. The fluorescence increased by a factor of 2 between −150 mV and+150 mV, with a nearly linear response throughout this range (FIG. 6D).The response of fluorescence to a step in membrane potential occurredwithin the 500 μs time resolution of our imaging system on both therising and falling edge (FIG. 6E). Application of a sinusoidally varyingmembrane potential led to sinusoidally varying fluorescence; at f=1 kHz,the fluorescence oscillations retained 55% of their low-frequencyamplitude (FIG. 18). Arch 3 reported voltage steps as small as 10 mV,with an accuracy of 625 μV/(Hz)^(1/2) over timescales <12 s (FIG. 19).

We tested Arch 3 as a voltage indicator in cultured rat hippocampalneurons, using viral delivery. Neurons expressing Arch 3 showedvoltage-dependent changes in fluorescence localized to the cellmembrane. Under whole cell current clamp, cells exhibited spiking uponinjection of current pulses of 200 pA. Individual spikes wereaccompanied by clearly identifiable increases of fluorescence (FIG. 7A).At a 2 kHz image acquisition rate, the signal-to-noise ratio in thefluorescence (spike amplitude:baseline noise) was 10.5. A spike-findingalgorithm correctly identified 99.6% of the spikes (based on comparisonto simultaneously recorded membrane potential), with a false-positiverate of 0.7% (n=269 spikes) (FIG. 7B). Single cells were observed for upto 4 minutes of cumulative exposure, with no detectable change inresting potential or spike frequency.

We imaged the dynamics of action potentials with sub-cellular resolutionby averaging multiple temporally registered movies of single spikes(FIG. 7C). In and near the soma, the optically determined waveform ofthe action potential was uniform and matched the electrically recordedwaveform. However in very thin processes the peak of the actionpotential lagged by up to 1 ms (FIG. 7D). These observations areconsistent with multiple-patch recordings on single neurons (Stuart, G.J. & Sakmann, B Active propagation of somatic action potentials intoneocortical pyramidal cell dendrites. Nature 367, 69-72 (1994)); butsuch recordings are technically demanding and only probe the variationin membrane potential at a small number of points. We show that Arch 3may be used to map intracellular dynamics of action potentials.Similarly, other archaerhodopsins can be expected to work in aeukaryotic cell as membrane potential indicators.

Thus, using a model system of Arch 3, we showed that the membranepotential of a mammalian cell can be detected using archaerhodopsins andmodified archaerhodopsins.

Gene Delivery Methods

The nucleic acids encoding the microbial rhodopsin proteins of theinvention are introduced to the cell or organ or organism of interestuding routine gene delivery methods. are administered to a subject forthe purpose of imaging membrane potential changes in cells of a subject.In one embodiment, the optical sensors are introduced to the cell viaexpression vectors.

The various gene delivery methods currently being applied to stem cellengineering include viral and non viral vectors, as well as biologicalor chemical methods of transfection. The methods can yield either stableor transient gene expression in the system used.

Viral Gene Delivery Systems

Because of their high efficiency of transfection, genetically modifiedviruses have been widely applied for the delivery of genes into stemcells.

DNA Virus Vectors

(i) Adenovirus

Adenoviruses are double stranded, nonenveloped and icosahedral virusescontaining a 36 kb viral genome (Kojaoghlanian et al., 2003). Theirgenes are divided into early (E1A, E1B, E2, E3, E4), delayed (IX, IVa2)and major late (L1, L2, L3, L4, L5) genes depending on whether theirexpression occurs before or after DNA replication. More than 51 humanadenovirus serotypes have been described which can infect and replicatein a wide range of organs. The viruses are classified into the followingsubgroups: A—induces tumor with high frequency and short latency, B—areweakly oncogenic, and C—are non-oncogenic (Cao et al., 2004;Kojaoghlanian et al., 2003).

These viruses have been used to generate a series of vectors for genetransfer cellular engineering. The initial generation of adenovirusvectors were produced by deleting the E1 gene (required for viralreplication) generating a vector with a 4 kb cloning capacity. Anadditional deletion of E3 (responsible for host immune response) allowedan 8 kb cloning capacity (Bett et al., 1994; Danthinne and Imperiale,2000; Danthinne and Werth, 2000). The second generation of vectors wasproduced by deleting the E2 region (required for viral replication)and/or the E4 region (participating in inhibition of host cellapoptosis) in conjunction with E1 or E3 deletions. The resultant vectorshave a cloning capacity of 10-13 kb (Armentano et al., 1995). The third“gutted” generation of vectors was produced by deletion of the entireviral sequence with the exception of the inverted terminal repeats(ITRs) and the cis acting packaging signals. These vectors have acloning capacity of 25 kb (Kochanek et al., 2001) and have retainedtheir high transfection efficiency both in quiescent and dividing cells.

Importantly, the adenovirus vectors do not normally integrate into thegenome of the host cell, but they have shown efficacy for transient genedelivery into adult stem cells. These vectors have a series ofadvantages and disadvantages. An important advantage is that they can beamplified at high titers and can infect a wide range of cells (Benihoudet al., 1999; Kanerva and Hemminki, 2005). The vectors are generallyeasy to handle due to their stability in various storing conditions.Adenovirus type 5 (Ad5) has been successfully used in delivering genesin human and mouse stem cells (Smith-Arica et al., 2003). The lack ofadenovirus integration into host cell genetic material can in manyinstances be seen as a disadvantage, as its use allows only transientexpression of the therapeutic gene.

The following provides examples to show that a skilled artisan canreadily transducer cells with contructs expressing microbial rhodopsinsof the present invention to eukaryotic, such as mammalian cells. Forexample in a study evaluating the capacity of mesenchymal stem cells toundergo chondrogenesis when TGF-beta1 and bone morphogencic protein-2(BMP-2) were delivered by adenoviral-mediated expression, thechondrogenesis was found to closely correlated with the level andduration of the transiently expressed proteins. Transgene expression inall aggregates was highly transient, showing a marked decrease after 7days. Chondrogenesis was inhibited in aggregates modified toexpress >100 ng/ml TGF-beta1 or BMP-2; however, this was partly due tothe inhibitory effect of exposure to high adenoviral loads (Mol. Ther.2005 August; 12 (2):219-28. Gene-induced chondrogenesis of primarymesenchymal stem cells in vitro. Palmer G D, Steinert A, Pascher A,Gouze E, Gouze J N, Betz O, Johnstone B, Evans C H, Ghivizzani S C). Ina second model using rat adipose derived stem cells transduced withadenovirus carrying the recombinant human bone morphogenic protein-7(BMP-7) gene showed promising results for an autologous source of stemcells for BMP gene therapy. However, activity assessed by measuringalkaline phosphatase in vitro was transient and peaked on day 8. Thusthe results were similar to those found in the chondrogenesis model(Cytotherapy. 2005; 7 (3):273-81).

Thus for experiments that do not require stable gene expressionadenovirus vectors is a good option.

Adenovirus vectors based on Ad type 5 have been shown to efficiently andtransiently introduce an exogenous gene via the primary receptor,coxsackievirus, and adenovirus receptor (CAR). However, some kinds ofstem cells, such as MSC and hematopoietic stem cells, cannot beefficiently transduced with conventional adenovirus vectors based on Adserotype 5 (Ad5), because of the lack of CAR expression. To overcomethis problem, fiber-modified adenovirus vectors and an adenovirus vectorbased on another serotype of adenovirus have been developed. (Mol.Pharm. 2006 March-April; 3 (2):95-103. Adenovirus vector-mediated genetransfer into stem cells. Kawabata K, Sakurai F, Koizumi N, Hayakawa T,Mizuguchi H. Laboratory of Gene Transfer and Regulation, NationalInstitute of Biomedical Innovation, Osaka 567-0085, Japan).

Such modifications can be readily applied to the use of the microbialrhodopsin constructs described herein, particularly in the applicationsrelating to stem cells.

(ii) Adeno-Associated Virus

Adeno-Associated viruses (AAV) are ubiquitous, noncytopathic,replication-incompetent members of ssDNA animal virus of parvoviridaefamily (G. Gao et al., New recombinant serotypes of AAV vectors. CurrGene Ther. 2005 June; 5 (3):285-97). AAV is a small icosahedral viruswith a 4.7 kb genome. These viruses have a characteristic terminiconsisting of palindromic repeats that fold into a hairpin. Theyreplicate with the help of helper virus, which are usually one of themany serotypes of adenovirus. In the absence of helper virus theyintegrate into the human genome at a specific locus (AAVS1) onchromosome 19 and persist in latent form until helper virus infectionoccurs (Atchison et al., 1965, 1966). AAV can transduce cell types fromdifferent species including mouse, rat and monkey. Among the serotypes,AAV2 is the most studied and widely applied as a gene delivery vector.Its genome encodes two large opening reading frames (ORFs) rep and cap.The rep gene encodes four proteins Rep 78, Rep 68, Rep 52 and Rep 40which play important roles in various stages of the viral life cycle(e.g. DNA replication, transcriptional control, site specificintegration, accumulation of single stranded genome used for viralpackaging). The cap gene encodes three viral capsid proteins VP1, VP2,VP3 (Becerra et al., 1988; Buning et al., 2003). The genomic 3′ endserves as the primer for the second strand synthesis and has terminalresolution sites (TRS) which serve as the integration sequence for thevirus as the sequence is identical to the sequence on chromosome 19(Young and Samulski, 2001; Young et al., 2000).

These viruses are similar to adenoviruses in that they are able toinfect a wide range of dividing and non-dividing cells. Unlikeadenovirus, they have the ability to integrate into the host genome at aspecific site in the human genome. Unfortunately, due to their ratherbulky genome, the AAV vectors have a limited capacity for the transferof foreign gene inserts (Wu and Ataai, 2000).

RNA Virus Vectors

(i) Retroviruses

Retroviral genomes consist of two identical copies of single strandedpositive sense RNAs, 7-10 kb in length coding for three genes; gag, poland env, flanked by long terminal repeats (LTR) (Yu and Schaffer, 2005).The gag gene encodes the core protein capsid containing matrix andnucleocapsid elements that are cleavage products of the gag precursorprotein. The pol gene codes for the viral protease, reversetranscriptase and integrase enzymes derived from gag-pol precursor gene.The env gene encodes the envelop glycoprotein which mediates viralentry. An important feature of the retroviral genome is the presence ofLTRs at each end of the genome. These sequences facilitate theinitiation of viral DNA synthesis, moderate integration of the proviralDNA into the host genome, and act as promoters in regulation of viralgene transcription. Retroviruses are subdivided into three generalgroups: the oncoretroviruses (Maloney Murine Leukenmia Virus, MoMLV),the lentiviruses (HIV), and the spumaviruses (foamy virus) (Trowbridgeet al., 2002).

Retroviral based vectors are the most commonly used integrating vectorsfor gene therapy. These vectors generally have a cloning capacity ofapproximately 8 kb and are generated by a complete deletion of the viralsequence with the exception of the LTRs and the cis acting packagingsignals.

The retroviral vectors integrate at random sites in the genome. Theproblems associated with this include potential insertional mutagenesis,and potential oncogenic activity driven from the LTR. The U3 region ofthe LTR harbors promoter and enhancer elements, hence this region whendeleted from the vector leads to a self-inactivating vector where LTRdriven transcription is prevented. An internal promoter can then be usedto drive expression of the transgene.

The initial studies of stem cell gene transfer in mice raised the hopethat gene transfer into humans would be equally as efficient (O'Connorand Crystal, 2006). Gene transfer using available retroviral vectorsystems to transfect multi-lineage long-term repopulating stem cells isstill significantly more efficient in the mouse.

(ii) Lentivirus

Lentiviruses are members of Retroviridae family of viruses (M. Scherr etal., Gene transfer into hematopoietic stem cells using lentiviralvectors. Curr Gene Ther. 2002 February; 2 (1):45-55). They have a morecomplex genome and replication cycle as compared to the oncoretroviruses(Beyer et al., 2002). They differ from simpler retroviruses in that theypossess additional regulatory genes and elements, such as the taat gene,which mediates the transactivation of viral transcription (Sodroski etal., 1996) and rev, which mediates nuclear export of unspliced viral RNA(Cochrane et al., 1990; Emerman and Temin, 1986).

Lentivirus vectors are derived from the human immunodeficiency virus(HIV-1) by removing the genes necessary for viral replication renderingthe virus inert. Although they are devoid of replication genes, thevector can still efficiently integrate into the host genome allowingstable expression of the transgene. These vectors have the additionaladvantage of a low cytotoxicity and an ability to infect diverse celltypes. Lentiviral vectors have also been developed from Simian, Equineand Feline origin but the vectors derived from Human ImmunodeficiencyVirus (HIV) are the most common (Young et al., 2006).

Lentivirus vectors are generated by deletion of the entire viralsequence with the exception of the LTRs and cis acting packagingsignals. The resultant vectors have a cloning capacity of about 8 kb.One distinguishing feature of these vectors from retroviral vectors istheir ability to transduce dividing and non-dividing cells as well asterminally differentiated cells (Kosaka et al., 2004). The lentiviraldelivery system is capable of high infection rates in human mesenchymaland embryonic stem cells. In a study by Clements et al., the lentiviralbackbone was modified to express mono- and bi-cistronic transgenes andwas also used to deliver short hairpin ribonucleic acid for specificsilencing of gene expression in human stem cells. (Tissue Eng. 2006July; 12 (7):1741-51. Lentiviral manipulation of gene expression inhuman adult and embryonic stem cells. Clements M O, Godfrey A, CrossleyJ, Wilson S J, Takeuchi Y, Boshoff C).

Table below summarizes some of the qualities of the viral vectors.

Insert Vector Vector capacity (kb) Tropism genome form ExpressionEfficiency Enveloped Retrovirus 8 Dividing cells Integrated Stable Highonly Lentivirus 8 Dividing and Integrated Stable High non-dividingNon-enveloped Adeno- <5 Dividing and Episomal and Stable High associatedvirus non-dividing integrated Adenovirus 2-24 Dividing and EpisomalTransient High non-dividingNon-Viral Gene Delivery Systems(i) Methods for the Facilitated Integration of Genes

In addition to the viral based vectors discussed above, other vectorsystems that lack viral sequence can be used. The alternative strategiesinclude conventional plasmid transfer and the application of targetedgene integration through the use of integrase or transposasetechnologies. These represent important new approaches for vectorintegration and have the advantage of being both efficient, and oftensite specific in their integration. Currently three recombinase systemsare available for genetic engineering: cre recombinase from phage P1(Lakso et al., 1992; Orban et al., 1992), FLP (flippase) from yeast 2micron plasmid (Dymecki, 1996; Rodriguez et al., 2000), and an integraseisolated from streptomyses phage I C31 (Ginsburg and Calos, 2005). Eachof these recombinases recognize specific target integration sites. Creand FLP recombinase catalyze integration at a 34 bp palindromic sequencecalled lox P (locus for crossover) and FRT (FLP recombinase target)respectively. Phage integrase catalyzes site-specific, unidirectionalrecombination between two short att recognition sites in mammaliangenomes. Recombination results in integration when the att sites arepresent on two different DNA molecules and deletion or inversion whenthe att sites are on the same molecule. It has been found to function intissue culture cells (in vitro) as well as in mice (in vivo).

The Sleeping Beauty (SB) transposon is comprised of two invertedterminal repeats of 340 base pairs each (Izsvak et al., 2000). Thissystem directs the precise transfer of specific constructs from a donorplasmid into a mammalian chromosome. The excision and integration of thetransposon from a plasmid vector into a chromosomal site is mediated bythe SB transposase, which can be delivered to cells as either in a cisor trans manner (Kaminski et al., 2002). A gene in a chromosomallyintegrated transposon can be expressed over the lifetime of a cell. SBtransposons integrate randomly at TA-dinucleotide base pairs althoughthe flanking sequences can influence integration.

Physical Methods to Introduce Vectors into Cells

(i) Electroporation

Electroporation relies on the use of brief, high voltage electric pulseswhich create transient pores in the membrane by overcoming itscapacitance. One advantage of this method is that it can be utilized forboth stable and transient gene expression in most cell types. Thetechnology relies on the relatively weak nature of the hydrophobic andhydrophilic interactions in the phospholipid membrane and its ability torecover its original state after the disturbance. Once the membrane ispermeabilized, polar molecules can be delivered into the cell with highefficiency. Large charged molecules like DNA and RNA move into the cellthrough a process driven by their electrophoretic gradient. Theamplitude of the pulse governs the total area that would bepermeabilized on the cell surface and the duration of the pulsedetermines the extent of permeabilization (Gabriel and Teissie, 1997).The permeabilized state of the cell depends on the strength of thepulses. Strong pulses can lead to irreversible permeabilization,irreparable damage to the cell and ultimately cell death. For thisreason electroporation is probably the harshest of gene delivery methodsand it generally requires greater quantities of DNA and cells. Theeffectiveness of this method depends on many crucial factors like thesize of the cell, replication and temperature during the application ofpulse (Rols and Teissie, 1990).

The most advantageous feature of this technique is that DNA can betransferred directly into the nucleus increasing its likelihood of beingintegrated into the host genome. Even cells difficult to transfect canbe stably transfected using this method (Aluigi et al., 2005; Zerneckeet al., 2003). Modification of the transfection procedure used duringelectroporation has led to the development of an efficient gene transfermethod called nucleofection. The Nucleofector™ technology, is anon-viral electroporation-based gene transfer technique that has beenproven to be an efficient tool for transfecting hard-to-transfect celllines and primary cells including MSC (Michela Aluigi, Stem Cells Vol.24, No. 2, February 2006, pp. 454-461).

Biomolecule-Based Methods

(i) Protein Transduction Domains (PTD)

PTD are short peptides that are transported into the cell without theuse of the endocytotic pathway or protein channels. The mechanisminvolved in their entry is not well understood, but it can occur even atlow temperature (Derossi et al. 1996). The two most commonly usednaturally occurring PTDs are the trans-activating activator oftranscription domain (TAT) of human immunodeficiency virus and thehomeodomain of Antennapedia transcription factor. In addition to thesenaturally occurring PTDs, there are a number of artificial peptides thathave the ability to spontaneously cross the cell membrane (Joliot andProchiantz, 2004). These peptides can be covalently linked to thepseudo-peptide backbone of PNA (peptide nucleic acids) to help deliverthem into the cell.

(ii) Liposomes

Liposomes are synthetic vesicles that resemble the cell membrane. Whenlipid molecules are agitated with water they spontaneously formspherical double membrane compartments surrounding an aqueous centerforming liposomes. They can fuse with cells and allow the transfer of“packaged” material into the cell. Liposomes have been successfully usedto deliver genes, drugs, reporter proteins and other biomolecules intocells (Felnerova et al., 2004). The advantage of liposomes is that theyare made of natural biomolecules (lipids) and are nonimmunogenic.

Diverse hydrophilic molecules can be incorporated into them duringformation. For example, when lipids with positively charged head groupare mixed with recombinant DNA they can form lipoplexes in which thenegatively charged DNA is complexed with the positive head groups oflipid molecules. These complexes can then enter the cell through theendocytotic pathway and deliver the DNA into lysosomal compartments. TheDNA molecules can escape this compartment with the help ofdioleoylethanolamine (DOPE) and are transported into the nucleus wherethey can be transcribed (Tranchant et al., 2004).

Despite their simplicity, liposomes suffer from low efficiency oftransfection because they are rapidly cleared by the reticuloendothelialsystem due to adsorption of plasma proteins. Many methods of stabilizingliposomes have been used including modification of the liposomal surfacewith oligosaccharides, thereby sterically stabilizing the liposomes (Xuet al., 2002).

(iii) Immunoliposomes

Immunoliposomes are liposomes with specific antibodies inserted intotheir membranes. The antibodies bind selectively to specific surfacemolecules on the target cell to facilitate uptake. The surface moleculestargeted by the antibodies are those that are preferably internalized bythe cells so that upon binding, the whole complex is taken up. Thisapproach increases the efficiency of transfection by enhancing theintracellular release of liposomal components. These antibodies can beinserted in the liposomal surface through various lipid anchors orattached at the terminus of polyethylene glycol grafted onto theliposomal surface. In addition to providing specificity to genedelivery, the antibodies can also provide a protective covering to theliposomes that helps to limit their degradation after uptake byendogenous RNAses or proteinases (Bendas, 2001). To further preventdegradation of liposomes and their contents in the lysosomalcompartment, pH sensitive immunoliposomes can be employed (Torchilin,2006). These liposomes enhance the release of liposomal content into thecytosol by fusing with the endosomal membrane within the organelle asthey become destabilized and prone to fusion at acidic pH.

In general non-viral gene delivery systems have not been as widelyapplied as a means of gene delivery into stem cells as viral genedelivery systems. However, promising results were demonstrated in astudy looking at the transfection viability, proliferation anddifferentiation of adult neural stem/progenitor cells into the threeneural lineages neurons. Non-viral, non-liposomal gene delivery systems(ExGen500 and FuGene6) had a transfection efficiency of between 16%(ExGen500) and 11% (FuGene6) of cells. FuGene6-treated cells did notdiffer from untransfected cells in their viability or rate ofproliferation, whereas these characteristics were significantly reducedfollowing ExGen500 transfection. Importantly, neither agent affected thepattern of differentiation following transfection. Both agents could beused to genetically label cells, and track their differentiation intothe three neural lineages, after grafting onto ex vivo organotypichippocampal slice cultures (J Gene Med. 2006 January; 8 (1):72-81.Efficient non-viral transfection of adult neural stem/progenitor cells,without affecting viability, proliferation or differentiation. Tinsley RB, Faijerson J, Eriksson P S).

(iv) Polymer-Based Methods

The protonated epsilon-amino groups of poly L-lysine (PLL) interact withthe negatively charged DNA molecules to form complexes that can be usedfor gene delivery. These complexes can be rather unstable and showed atendency to aggregate (Kwoh et al., 1999). The conjugation ofpolyethylene glycol (PEG) was found to lead to an increased stability ofthe complexes (Lee et al., 2005, Harada-Shiba et al., 2002). To confer adegree of tissue-specificity, targeting molecules such astissue-specific antibodies have also been employed (Trubetskoy et al.,1992, Suh et al., 2001).

An additional gene carrier that has been used for transfecting cells ispolyethylenimine (PEI) which also forms complexes with DNA. Due to thepresence of amines with different pKa values, it has the ability toescape the endosomal compartment (Boussif et al., 1995). PEG graftedonto PEI complexes was found to reduce the cytotoxicity and aggregationof these complexes. This can also be used in combination with conjugatedantibodies to confer tissue-specificity (Mishra et al., 2004, Shi etal., 2003, Chiu et al., 2004, Merdan et al., 2003).

Targeted Gene Delivery—Site-specific Recombinations

In certain embodiments, a non-human, transgenic animal comprising atargeting vector that further comprises recombination sites (e.g., Loxsites, FRT sites) can be crossed with a non-human, transgenic animalcomprising a recombinase (e.g., Cre recombinase, FLP recombinase) undercontrol of a particular promoter. It has been shown that thesesite-specific recombination systems, although of microbial origin forthe majority, function in higher eukaryotes, such as plants, insects andmice. Among the site-specific recombination systems commonly used, theremay be mentioned the Cre/Lox and FLP/FRT systems. The strategy normallyused consists of inserting the loxP (or FRT) sites into the chromosomesof ES cells by homologous recombination, or by conventionaltransgenesis, and then of delivering Cre (or FLP) for the latter tocatalyze the recombination reaction. The recombination between the twoloxP (or FRT) sites may be obtained in ES cells or in fertilized eggs bytransient expression of Cre or using a Cre transgenic mouse. Such astrategy of somatic mutagenesis allows a spatial control of therecombination because the expression of the recombinase is controlled bya promoter specific for a given tissue or for a given cell.

A detailed description of the FRT system can be found, e.g., in U.S.Pat. No. 7,736,897.

The P1 bacteriophage uses Cre-lox recombination to circularize andfacilitate replication of its genomic DNA when reproducing. Since beingdiscovered, the bacteriophage's recombination strategy has beendeveloped as a technology for genome manipulation. Because the cre geneand loxP sites are not native to the mouse genome, they are introducedby transgenic technology into the mouse genomes (Nagy A. 2000. Crerecombinase: the universal reagent for genome tailoring. Genesis26:99-109). The orientation and location of the loxP sites determinewhether Cre recombination induces a deletion, inversion, or chromosomaltranslocation (Nagy A. 2000. Cre recombinase: the universal reagent forgenome tailoring. Genesis 26:99-109). The cre/lox system has beensuccessfully applied in mammalian cell cultures, yeasts, plants, mice,and other organisms (Araki K, Imaizumi T, Okuyama K, Oike Y, Yamamura K.1997. Efficiency of recombination by Cre transient expression inembryonic stem cells: comparison of various promoters. J Biochem (Tokyo)122:977-82). Much of the success of Cre-lox is due to its simplicity. Itrequires only two components: (a) Cre recombinase: an enzyme thatcatalyzes recombination between two loxP sites; and (b) LoxP sites: aspecific 34-base pair bp) sequences consisting of an 8-bp core sequence,where recombination takes place, and two flanking 13-bp invertedrepeats.

Cell-mediated Delivery

In one embodiment, the optical sensors of the present invention aredelivered using e.g., a cell expressing the optical sensor. A variety ofmeans for administering cells to subjects are known to those of skill inthe art. Such methods can include systemic injection, for example i.v.injection or implantation of cells into a target site in a subject.Cells may be inserted into a delivery device which facilitatesintroduction by injection or implantation into the subjects. Suchdelivery devices may include tubes, e.g., catheters, for injecting cellsand fluids into the body of a recipient subject. In one preferredembodiment, the tubes additionally have a needle, e.g., a syringe,through which the cells of the invention can be introduced into thesubject at a desired location. The cells may be prepared for delivery ina variety of different forms. For example, the cells may be suspended ina solution or gel or embedded in a support matrix when contained in sucha delivery device. Cells may be mixed with a pharmaceutically acceptablecarrier or diluent in which the cells of the invention remain viable.Pharmaceutically acceptable carriers and diluents include saline,aqueous buffer solutions, solvents and/or dispersion media. The use ofsuch carriers and diluents is well known in the art. The solution ispreferably sterile and fluid. Preferably, the solution is stable underthe conditions of manufacture and storage and preserved against thecontaminating action of microorganisms such as bacteria and fungithrough the use of, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. Solutions of the invention maybe prepared by incorporating cells as described herein in apharmaceutically acceptable carrier or diluent and, as required, otheringredients enumerated above, followed by filtered sterilization. It ispreferred that the mode of cell administration is relativelynon-invasive, for example by intravenous injection, pulmonary deliverythrough inhalation, oral delivery, buccal, rectal, vaginal, topical, orintranasal administration.

However, the route of cell administration will depend on the tissue tobe treated and may include implantation or direct injection. Methods forcell delivery are known to those of skill in the art and can beextrapolated by one skilled in the art of medicine for use with themethods and compositions described herein. Direct injection techniquesfor cell administration can also be used to stimulate transmigrationthrough the entire vasculature, or to the vasculature of a particularorgan, such as for example liver, or kidney or any other organ. Thisincludes non-specific targeting of the vasculature. One can target anyorgan by selecting a specific injection site, such as e.g., a liverportal vein. Alternatively, the injection can be performed systemicallyinto any vein in the body. This method is useful for enhancing stem cellnumbers in aging patients. In addition, the cells can function topopulate vacant stem cell niches or create new stem cells to replenishthe organ, thus improving organ function. For example, cells may take uppericyte locations within the vasculature. Delivery of cells may also beused to target sites of active angiogenesis. If so desired, a mammal orsubject can be pre-treated with an agent, for example an agent isadministered to enhance cell targeting to a tissue (e.g., a homingfactor) and can be placed at that site to encourage cells to target thedesired tissue. For example, direct injection of homing factors into atissue can be performed prior to systemic delivery of ligand-targetedcells.

Method of using stem cells, such as neural stem cells to deliver agentsthrough systemic administration and via intracranial administration tohome in on a tumor or to an injured parts of brain have been described(see, e.g., U.S. Pat. Nos. 7,655,224; and 7,393,526). Accordingly, onecan also modify such cells to express the desired voltage sensor fordelivery into the organs, such as the brain.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, references to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

It is understood that the foregoing detailed description and thefollowing examples are illustrative only and are not to be taken aslimitations upon the scope of the invention. Various changes andmodifications to the disclosed embodiments, which will be apparent tothose skilled in the art, may be made without departing from the spiritand scope of the present invention. Further, all patents, patentapplications, and publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments are based on the information available to the applicants anddo not constitute any admission as to the correctness of the dates orcontents of these documents.

Some Definitions

As used herein the term “optical sensor” refers to a microbial rhodopsinprotein employed to yield an optical signal indicative of the voltagedrop across the membrane in which it is embedded

As used herein the phrase “reduced ion pumping activity” means adecrease in the endogenous ion pumping activity of a modified microbialrhodopsin protein of at least 10% compared to the endogenous pumpingactivity of the natural microbial rhodopsin protein from which themodified rhodopsin is derived. The ions most commonly pumped bymicrobial rhodopsins are H⁺ and Cl⁻. In some embodiments, the ionpumping activity of a modified rhodopsin protein is at least 20% lower,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, or at least 99% lower than theendogenous ion pumping activity of the corresponding native microbialrhodopsin protein.

In some embodiments, the modified microbial rhodopsin has no detectableion pumping activity.

As used herein, the term “endogenous ion pumping activity” refers to themovement of ions through a native microbial rhodopsin protein thatoccurs in response to light stimuli.

As used herein, the term “native microbial rhodopsin protein” or“natural microbial rhodopsin protein” refers to a rhodopsin proteinprepared or isolated from a microbial (e.g., bacterial, archaeal, oreukaryotic) source. Such natural microbial rhodopsin proteins, whenisolated, retain characteristics (e.g., pKa, ion pumping activity etc)that are substantially similar to the microbial rhodopsin protein in itsnative environment (e.g., in a microbial cell). Some non-limitingexamples of microbial rhodopsin proteins useful with the methodsdescribed herein include green-absorbing proteorhodopsin (GPR; GenBankaccession number AF349983), blue-absorbing proteorhodopsin (BPR, GenBankaccession number AF349981), Natromonas pharaonis sensory rhodopsin II(NpSRII; GenBank accession number Z35086.1), and bacteriorhodopsin (BR;the protein encoded by GenBank sequence NC_010364.1, nucleotides1082241-1083029, wherein 1082241 is designated as 1 herein, GenBankaccession number M11720.1, or as described by e.g., Beja O, et al.,(2000). Science 289 (5486): 1902-1904), and archaerhodopsin (see e.g.,Chow B. Y. et al., Nature 463:98-102 (2010) and the Examples in thisapplication).

As used herein, the term “modified microbial rhodopsin protein” refersto a native microbial rhodopsin protein comprising at least onemutation. Mutations can be in the nucleic acid sequence (e.g., genomicor mRNA sequence), or alternatively can comprise an amino acidsubstitution. Such amino acid substitutions can be conserved mutationsor non-conserved mutations. As well-known in the art, a “conservativesubstitution” of an amino acid or a “conservative substitution variant”of a polypeptide refers to an amino acid substitution whichmaintains: 1) the structure of the backbone of the polypeptide (e.g. abeta sheet or alpha-helical structure); 2) the charge or hydrophobicityof the amino acid; or 3) the bulkiness of the side chain. Morespecifically, the well-known terminologies “hydrophilic residues” relateto serine or threonine. “Hydrophobic residues” refer to leucine,isoleucine, phenylalanine, valine or alanine. “Positively chargedresidues” relate to lysine, arginine or histidine. “Negatively chargedresidues” refer to aspartic acid or glutamic acid. Residues having“bulky side chains” refer to phenylalanine, tryptophan or tyrosine. Toavoid doubt as to nomenclature, the term “D97N” or similar termsspecifying other specific amino acid substitutions means that the Asp(D) at position 97 of the protein sequence is substituted with Asn (N).A “conservative substitution variant” of D97N would substitute aconservative amino acid variant of Asn (N) that is not D.

The terminology “conservative amino acid substitutions” is well known inthe art, which relates to substitution of a particular amino acid by onehaving a similar characteristic (e.g., similar charge or hydrophobicity,similar bulkiness). Examples include aspartic acid for glutamic acid, orisoleucine for leucine. A list of exemplary conservative amino acidsubstitutions is given in the Table 5 below. A conservative substitutionmutant or variant will 1) have only conservative amino acidsubstitutions relative to the parent sequence, 2) will have at least 90%sequence identity with respect to the parent sequence, preferably atleast 95% identity, 96% identity, 97% identity, 98% identity or 99%; and3) will retain voltage sensing activity as that term is defined herein.

TABLE 5 Conservative Amino Acid Substitutions For Amino Acid CodeReplace With Alanine A D-ala, Gly, Aib, β-Ala, Acp, L-Cys, D-CysArginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile, D- Met,D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-GlnAspartic Acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine CD-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr Glutamine Q D-Gln, Asn, D-Asn,Glu, D-Glu, Asp, D-Asp Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn,Gln, D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, Aib, β-Ala, Acp IsoleucineI D-Ile, Val, D-Val, AdaA, AdaG, Leu, D-Leu, Met, D-Met Leucine L D-Leu,Val, D-Val, AdaA, AdaG, Leu, D-Leu, Met, D-Met Lysine K D-Lys, Arg,D-Arg, homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-OrnMethionine M D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-ValPhenylalanine F D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp,Trans- 3,4 or 5-phenylproline, AdaA, AdaG, cis-3,4 or 5- phenylproline,Bpa, D-Bpa Proline P D-Pro, L-I-thioazolidine-4-carboxylic acid,D-or-L-1- oxazolidine-4-carboxylic acid (Kauer, U.S. Pat. No.(4,511,390) Serine S D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met (O),D-Met (O), L-Cys, D-Cys Threonine T D-Thr, Ser, D-Ser, allo-Thr, Met,D-Met, Met (O), D-Met (O), Val, D-Val Tyrosine Y D-Tyr, Phe, D-Phe,L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met,AdaA, AdaG

A non-conservative mutation is any other amino acid substitution otherthan the conservative substitutions noted in the above table.

Methods of making conservative amino acid substitutions are also wellknown to one skilled in the art and include but are not limited tosite-specific mutagenesis using oligonucleotide primers and polymerasechain reactions. Optical sensor variants can be expressed and assayedfor voltage sensing activity, pKa, and fluorescence detection by methodsknown in the art and/or described herein to verify that the desiredactivities of the optical sensor are retained or augmented by the aminoacid substitutions. It is contemplated that conservative amino acidsubstitution variants of the optical sensors described herein can haveenhanced activity or superior characteristics for sensing voltagerelative to the parent optical sensor. Certain silent or neutralmissense mutations can also be made in the nucleic acid encoding anoptical sensor by a mutation that does not change the encoded amino acidsequence of the encoded optical sensor. These types of mutations areuseful to optimize codon usage which improve recombinant proteinexpression and production in the desired cell type. Specificsite-directed mutagenesis of a nucleic acid encoding an optical sensorin a vector can be used to create specific amino acid mutations andsubstitutions. Site-directed mutagenesis can be carried out using, e. g.the QUICKCHANGE® site-directed mutagenesis kit from STRATAGENE®according to manufacture's instructions, or by any method known in theart.

As used herein, the term “membrane potential” refers to a calculateddifference in voltage between the interior and exterior of a cell. Inone embodiment membrane potential, ΔV, is determined by the equationΔV=V_(interior)−V_(exterior). For example, if the outside voltage is 100mV, and the inside voltage is 30 mV, then the difference is −70 mV.Under resting conditions, the membrane potential is predominantlydetermined by the ion having the greatest conductance across themembrane. In many cells, the membrane potential is determined bypotassium, which yields a resting membrane potential of approximately−70 mV. Thus by convention, a cell under resting conditions has anegative membrane potential. In some cells when a membrane potential isreached that is equal to or greater than a threshold potential, anaction potential is triggered and the cell undergoes depolarization(i.e., a large increase in the membrane potential). Often, when a cellundergoes depolarization, the membrane potential reverses and reachespositive values (e.g., 55 mV). During resolution of the membranepotential following depolarization towards the resting membranepotential, a cell can “hyperpolarize.” The term “hyperpolarize” refersto membrane potentials that are more negative than the resting membranepotential, while the term “depolarize” refers to membrane potentialsthat are less negative (or even positive) compared to the restingmembrane potential. Membrane potential changes can arise by movement ofions through ion channels or ion pumps embedded in the membrane.Membrane potential can be measured across any cellular membrane thatcomprises ion channels or ion pumps that can maintain an ionic gradientacross the membrane (e.g., plasma membrane, mitochondrial inner andouter membranes etc.).

As used herein, the term “change in the membrane potential” refers to anincrease (or decrease) in ΔV of at least 1 mV that is either spontaneousor in response to e.g., environmental or chemical stimuli (e.g.,cell-to-cell communication, ion channel modulation, contact with acandidate agent etc.) compared to the resting membrane potentialmeasured under control conditions (e.g., absence of an agent, impairedcellular communication, etc.). In some embodiments, the membranepotential ΔV is increased by at least 10 mV, at least 15 mV, at least 20mV, at least 25 mV, at least 30 mV, at least 35 mV, at least 40 mV, atleast 45 mV, at least 50 mV, at least 55 mV, at least 60 mV, at least 65mV, at least 70 mV, at least 75 mV, at least 80 mV, at least 85 mV, atleast 90 mV, at least 95 mV, at least 100 mV, at least 105 mV, at least110 mV, at least 115 mV, at least 120 mV, at least 125 mV, at least 130mV, at least 135 mV, at least 140 mV, at least 145 mV, at least 150 mV,at least 155 mV, at least 160 mV, at least 165 V, at least 170 mV, atleast 180 mV, at least 190 mV, at least 200 mV or more compared to themembrane potential of a similar cell under control conditions. In otherembodiments, the membrane potential is decreased by at least 3 mV, atleast 5 mV, at least 10 mV, at least 15 mV, at least 20 mV, at least 25mV, at least 30 mV, at least 35 mV, at least 40 mV, at least 45 mV, atleast 50 mV, at least 55 mV, at least 60 mV, at least 65 mV, at least 70mV, at least 75 mV, at least 80 mV, at least 85 mV, at least 90 mV, atleast 95 mV, at least 100 mV, at least 105 mV, at least 110 mV, at least115 mV, at least 120 mV, at least 125 mV, at least 130 mV, at least 135mV, at least 140 mV, at least 145 mV, at least 150 mV or more comparedto the membrane potential of a similar cell under control conditions.

As used herein, the phrase “localizes to a membrane of the cell” refersto the preferential localization of the modified microbial rhodopsinprotein to the membrane of a cell and can be achieved by e.g., modifyingthe microbial rhodopsin to comprise a signal sequence that directs therhodopsin protein to a membrane of the cell (e.g., the plasma membrane,the mitochondrial outer membrane, the mitochondrial inner membraneetc.). In some embodiments, at least 40% of the modified microbialrhodopsin protein in the cell is localized to the desired cellularmembrane compartment (e.g., plasma membrane, mitochondrial membraneetc); in other embodiments, at least 50%, at least 60%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 99% of the modified microbial rhodopsin protein is localized tothe desired cellular membrane compartment. Similarly, the phrase“localized to a subcellular compartment” refers to the preferentiallocalization of the microbial rhodopsin protein to a particularsubcellular compartment (e.g., mitochondria, endoplasmic reticulum,peroxisome etc.). In some embodiments, at least 40% of the modifiedmicrobial rhodopsin protein in the cell is localized to the desiredsubcellular compartment; in other embodiments, at least 50%, at least60%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or at least 99% of the modified microbial rhodopsinprotein is localized to the desired subcellular compartment.

In some embodiment, about 100% is localized to the desired cellularmembrane or compartment.

As used herein, the term “introducing to a cell” refers to any methodfor introducing either an expression vector encoding an optical sensoror a recombinant optical sensor protein described herein into a hostcell. Some non-limiting examples of introducing an expression vectorinto a cell include, for example, calcium phosphate transfection,electroporation, lipofection, or a method using a gene gun or the like.In one embodiment, a recombinant optical sensor protein is introduced toa cell by membrane fusion using a lipid mediated delivery system, suchas micelles, liposomes, etc.

As used herein, the phrase “a moiety that produces an optical signal”refers to a molecule (e.g., retinal), or moiety of a molecule, capableof producing a detectable signal such as e.g., fluorescence,chemiluminescence, a colorimetric signal etc. In one embodiment, themodified microbial rhodopsin comprises a fusion molecule with a moietythat produces an optical signal.

As used herein, the phrases “change in the level of fluorescence” or “achange in the level of the optical signal” refer to an increase ordecrease in the level of fluorescence from the modified microbialrhodopsin protein or an increase or decrease in the level of the opticalsignal induced by a change in voltage or membrane potential. In someembodiments, the level of fluorescence or level of optical signal in acell is increased by at least at least 2%, at least 5%, at least 10%,20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 99%, at least1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least50-fold, at least 100-fold, at least 500-fold, at least 600-fold, atleast 700-fold, at least 800-fold, at least 900-fold, at least1000-fold, at least 2000-fold, at least 5000-fold, at least 10000-foldor more compared to the same cell or a similar cell under controlconditions. Alternatively, the level of fluorescence or level of opticalsignal in a cell is decreased by at least by at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 99%, or even 100% (i.e., no detectablesignal) compared to the same cell or a similar cell under controlculture conditions.

As used herein, the phrase “modulates ion channel activity” refers to anincrease or decrease in one or more properties of an ion channel thatmanifests as a change in the membrane potential of a cell. Theseproperties include, e.g., open- or closed-state conductivity, thresholdvoltage, kinetics and/or ligand affinity. In some embodiments, the oneor more properties of interest of an ion channel of a cell as measuredby e.g., a change in membrane potential of the cell. In someembodiments, the activity of an ion channel is increased by at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 99%, at least1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or morein the presence of an agent compared to the activity of the ion channelin the absence of the agent. In other embodiments, the parameter ofinterest of an ion channel is decreased by at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, or at least 99% in the presence of an agentcompared to the activity of the ion channel in the absence of the agent.In some embodiments, the parameter of an ion channel is absent in thepresence of an agent compared to the activity of the ion channel in theabsence of the agent.

As used herein, the term “inhibitor of antibiotic efflux” refers to anagent that decreases the level of antibiotic efflux from a bacterialcell by at least 20% compared to the level of antibiotic efflux in theabsence of the agent. In some embodiments, antibiotic efflux isdecreased by at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 99%, oreven 100% (i.e., absent) in the presence of an agent compared to thelevel of antibiotic efflux in the absence of the agent.

As used herein, the term “targeting sequence” refers to a moiety orsequence that homes to or preferentially associates or binds to aparticular tissue, cell type, receptor, organelle, or other area ofinterest. The addition of a targeting sequence to an optical sensorcomposition will enhance the delivery of the composition to a desiredcell type or subcellular location. The addition to, or expression of, atargeting sequence with the optical sensor in a cell enhances thelocalization of the optical sensor to a desired location within ananimal or subject.

As used herein, the phrase “homologous mutation in another microbialrhodopsin that corresponds to the amino acid mutation inbacteriorhodopsin” refers to mutation of a residue in a desiredmicrobial rhodopsin that is expected to have a similar effect to asubstantially similar mutation in bacteriorhodopsin. One of skill in theart can easily locate a homologous residue in their desired microbialrhodopsin by performing an alignment of conserved regions of the desiredmicrobial rhodopsin with a bacteriorhodopsin sequence using a computerprogram such as ClustalW.

Examples of homologous mutations include the mutations made in theExamples set forth in this application.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

Provided herein are optical sensors comprising a microbial rhodopsin ora modified microbial rhodopsin protein with a reduced ion pumpingactivity compared to a natural microbial rhodopsin protein from which itis derived. In one embodiment, the composition comprises a vectorencoding or an engineered cell comprising the microbial rhodopsinprotein or the modified microbial rhodopsin protein with a reduced ionpumping activity compared to a natural microbial rhodopsin protein fromwhich it is derived. In some embodiments, the microbial rhodopsin isdirected to a membrane, such as plasma membrane or mitochondrialmembrane.

Accordingly, the invention provides a method for measuring membranepotential in a cell expressing a nucleic acid encoding a microbialrhodopsin protein, the method comprising the steps of: (a) exciting, invitro, ex vivo or in vivo, at least one cell comprising a nucleic acidencoding a microbial rhodopsin protein with light of at least one wavelength; and (b) detecting, in vitro, at least one optical signal fromthe at least one cell, wherein the level of fluorescence emitted by theat least one cell compared to a reference is indicative of the membranepotential of the cell.

In some or any embodiment or aspect of the invention, the he microbialrhodopsin protein is a modified microbial rhodopsin protein with reducedion pumping activity compared to a natural microbial rhodopsin proteinfrom which it is derived.

In some or any embodiment or aspect of the invention, the modifiedmicrobial rhodopsin comprises a mutated proton acceptor proximal to theSchiff Base.

In some or any embodiment or aspect of the invention, the microbialrhodopsin protein is a member of the proteorhodopsin family of proteins.

In some or any embodiment or aspect of the invention the microbialrhodopsin protein is a member of the archaerhodopsin family of proteins.

In some or any embodiment or aspect of the invention the at least onewave length is a wave length between λ=594-645 nm.

In some or any embodiment or aspect of the invention the cell is aprokaryotic cell.

In some or any embodiment or aspect of the invention the cell is aeukaryotic cell.

In some or any embodiment or aspect of the invention the eukaryotic cellis a mammalian cell.

In some or any embodiment or aspect of the invention the eukaryotic cellis a stem cell or a pluripotent or a progenitor cell.

In some or any embodiment or aspect of the invention the eukaryotic cellis an induced pluripotent cell.

In some or any embodiment or aspect of the invention the eukaryotic cellis a neuron.

In some or any embodiment or aspect of the invention the eukaryotic cellis a cardiomyocyte.

In some or any embodiment or aspect of the invention the method furthercomprises a step of transfecting, in vitro, ex vivo or in vivo, the atleast one cell with a vector comprising the nucleic acid encoding themicrobial rhodopsin protein.

In some or any embodiment or aspect of the invention, the nucleic acidencoding the microbial rhodopsin protein is operably linked to acell-type specific promoter.

In some or any embodiment or aspect of the invention, the nucleic acidencoding the microbial rhodopsin protein is operably linked to amembrane-targeting nucleic acid sequence.

In some or any embodiment or aspect of the invention, themembrane-targeting nucleic acid is a plasma membrane targeting nucleicacid sequence.

In some or any embodiment or aspect of the invention, themembrane-targeting nucleic acid sequence is a subcellularcompartment-targeting nucleic acid sequence.

In some or any embodiment or aspect of the invention, the subcellularcompartment is selected from a mitochondrial membrane, an endoplasmicreticulum, a sarcoplastic reticulum, a synaptic vesicle, an endosome anda phagosome.

In some or any embodiment or aspect of the invention, the nucleic acidencoding a microbial rhodopsin protein is operably linked to a nucleicacid encoding at least one additional fluorescent protein or achromophore.

In some or any embodiment or aspect of the invention, the at least oneadditional fluorescent protein is a fluorescent protein capable forindicating the ion concentration in the cell.

In some or any embodiment or aspect of the invention, the fluorescentprotein capable for indicating ion concentration is a calcium indicator.

In some or any embodiment or aspect of the invention, the fluorescentprotein capable for indicating ion concentration is a pH indicator.

In some or any embodiment or aspect of the invention, the at least oneadditional fluorescent protein is capable of undergoing nonradiativefluorescence resonance energy transfer to the microbial rhodopsin, witha rate of energy transfer dependent on the membrane potential.

In some or any embodiment or aspect of the invention, the at least oneadditional fluorescent protein is a green fluorescent protein or ahomolog thereof.

In some or any embodiment or aspect of the invention, brightness of thefluorescent protein is insensitive to membrane potential and localchemical environment.

In some or any embodiment or aspect of the invention, the method furthercomprises steps of exciting, in vitro, ex vivo or in vivo, the at leastone cell with light of at least a first and a second wavelength; anddetecting, in vitro, ex vivo or in vivo, the at least first and thesecond optical signal resulting from the excitation with the at leastthe first and the second wavelength from the at least one cell.

In some or any embodiment or aspect of the invention, the at leastsecond wave length is between λ=447-594 nm.

In some or any embodiment or aspect of the invention, the method furthercomprises a step of calculating the ratio of the fluorescence emissionfrom the microbial rhodopsin to the fluorescence emission of the atleast one additional fluorescent protein to obtain a measurement ofmembrane potential independent of variations in expression level.

In some or any embodiment or aspect of the invention, the method furthercomprises the step of exposing, in vitro, ex vivo or in vivo, the atleast one cell to a stimulus capable of, or suspected to be capable ofchanging membrane potential.

In some or any embodiment or aspect of the invention, the stimulus acandidate agent.

In some or any embodiment or aspect of the invention, the stimulus is achange to the composition of the cell culture medium.

In some or any embodiment or aspect of the invention, the stimulus is anelectrical current.

In some or any embodiment or aspect of the invention, further comprisingthe step of measuring, in vitro, ex vivo or in vivo the at least oneoptical signal at a first and at least at a second time point.

In some or any embodiment or aspect of the invention, the first timepoint is before exposing the at least one cell to a stimulus and the atleast second time point is after exposing the at least one cell to thestimulus.

In some or any embodiment or aspect of the invention, wherein the methodcomprises a plurality of cells, for example in a highthroughput assay.

In another embodiment, the invention provides an isolated and purifiednucleic acid encoding a modified member of the archaerhodopsin family ofproteins with reduced ion pumping activity compared to a natural memberof the archaerhodopsin family of proteins from which it is derived.

In some or any embodiment or aspect of the invention, the modifiedmember of an archaerhodopsin family of proteins comprises a mutatedproton acceptor proximal to the Schiff Base.

In some or any embodiment or aspect of the invention, the modifiedmember of the archaerhodopsin family of proteins with reduced ionpumping activity compared to a natural member of the archaerhodopsinfamily of proteins from which it is derived is operably linked to anucleic acid sequence encoding a membrane-targeting nucleic acidsequence.

In some or any embodiment or aspect of the invention, themembrane-targeting nucleic acid sequence is a plasma membrane targetingnucleic acid sequence.

In some or any embodiment or aspect of the invention, themembrane-targeting nucleic acid sequence is a subcellularmembrane-targeting nucleic acid sequence.

In some or any embodiment or aspect of the invention, the subcellularmembrane is a mitochondrial membrane, an endoplasmic reticulum, asarcoplastic reticulum, a synaptic vesicle, an endosome or a phagosome.

In some or any embodiment or aspect of the invention, the modifiedmicrobial rhodopsin of the archaerhodopsin family is operably linked toa cell-type specific promoter.

In some or any embodiment or aspect of the invention, the modifiedmicrobial rhodopsin of the archaerhodopsin family is operably linked toa nucleic acid encoding at least one additional fluorescent protein or achromophore.

In some or any embodiment or aspect of the invention, wherein the atleast one additional fluorescent protein is a green fluorescent proteinor a homolog thereof.

In some or any embodiment or aspect of the invention, the at least oneadditional fluorescent protein is a fluorescent protein capable forindicating ion concentration in the cell.

In some or any embodiment or aspect of the invention, the fluorescentprotein capable for indicating ion concentration is a calcium indicator.

In some or any embodiment or aspect of the invention, the fluorescentprotein capable for indicating ion concentration is a pH indicator.

In some or any embodiment or aspect of the invention, the at least oneadditional fluorescent protein is capable of undergoing nonradiativefluorescence resonance energy transfer to the microbial rhodopsin, witha rate of energy transfer dependent on the membrane potential.

In some or any embodiment or aspect of the invention, the isolated andpurified nucleic further comprises a vector.

In some or any embodiment or aspect of the invention, the vector is aviral vector, such as a lentiviral vector or an adeno-associated viralvector.

In another embodiment, the invention provides a kit comprising theisolated and purified nucleic acid encoding the modified microbialproteins of archaerhodopsin family as disclosed herein in a suitablecontainer. The nucleic acids may be provided in dry form or in asuitable buffer.

Another embodiment of the invention provides an isolated cell comprisinga nucleic acid encoding a microbial rhodopsin protein, wherein themicrobial rhodopsin protein is a modified microbial rhodopsin proteinwith reduced ion pumping activity compared to a natural microbialrhodopsin protein from which it is derived.

In some or any embodiment or aspect of the invention, the modifiedmicrobial rhodopsin protein comprises a mutated proton acceptor proximalto the Schiff Base.

In some or any embodiment or aspect of the invention, the microbialrhodopsin is a member of the proteorhodopsin family.

In some or any embodiment or aspect of the invention, the microbialrhodopsin is a member of the archaerhodopsin family.

In some or any embodiment or aspect of the invention, the cell is aeukaryotic cell.

In some or any embodiment or aspect of the invention, the cell is aprokaryotic cell.

In some or any embodiment or aspect of the invention, the modifiedmicrobial rhodopsin gene is operably linked to a promoter.

In some or any embodiment or aspect of the invention, the promoter is acell-type specific promoter.

In some or any embodiment or aspect of the invention, the nucleic acidencoding the modified microbial rhodopsin protein is operably linked toa membrane-targeting nucleic acid sequence.

In some or any embodiment or aspect of the invention, themembrane-targeting nucleic acid is a plasma membrane targeting nucleicacid sequence.

In some or any embodiment or aspect of the invention, themembrane-targeting nucleic acid is a subcellular compartment-targetingnucleic acid sequence.

In some or any embodiment or aspect of the invention, the subcellularcompartment is selected from a mitochondrial membrane, an endoplasmicreticulum, a sarcoplastic reticulum, a synaptic vesicle, an endosome anda phagosome.

In some or any embodiment or aspect of the invention, the nucleic acidencoding the modified microbial rhodopsin protein is operably linked toa nucleic acid encoding at least one additional fluorescent protein orchromophore.

In some or any embodiment or aspect of the invention, the at least oneadditional fluorescent protein is a green fluorescent protein or ahomolog thereof.

In some or any embodiment or aspect of the invention, the at least oneadditional fluorescent protein is capable of undergoing nonradiativefluorescence resonance energy transfer to the microbial rhodopsin, witha rate of energy transfer dependent on the membrane potential.

In some or any embodiment or aspect of the invention, the least oneadditional fluorescent protein is a fluorescent protein whose brightnessis insensitive to membrane potential and local chemical environment.

In some or any embodiment or aspect of the invention, the at least oneadditional fluorescent protein is capable of indicating the ionconcentration in the cell.

In some or any embodiment or aspect of the invention, the fluorescentprotein capable for indicating ion concentration is a calcium indicator.

In some or any embodiment or aspect of the invention, the fluorescentprotein capable for indicating ion concentration is a pH indicator.

In some or any embodiment or aspect of the invention, the cell is a stemcell, a pluripotent cell, or an induced pluripotent cell, ordifferentiated or undifferentiated progeny thereof.

In some or any embodiment or aspect of the invention, the cell is adifferentiated cell.

In some or any embodiment or aspect of the invention, the differentiatedcell is a neuron.

In some or any embodiment or aspect of the invention, the differentiatedcell is a cardiomyocyte.

The invention further provides, in one embodiment, an isolated cellcomprising the isolated and purified nucleic acid of any of the isolatedand purified nucleic acids or nucleic acid constructs comprising themodified microbial rhodopsin of archaerhodopsin family as describedabove.

In one embodiment, the invention provides a kit comprising a pluralityof cells as described herein in a suitable cell culture medium and acontainer.

In yet another embodiment, the invention provides a method of making anengineered cell for optical measurement of membrane potential comprisingthe steps of transducing a cell with a nucleic acid encoding a microbialrhodopsin protein.

In some or any embodiment or aspect of the invention, the microbialrhodopsin protein is a modified microbial rhodopsin protein with reducedion pumping activity compared to a natural microbial rhodopsin protein.

In some or any embodiment or aspect of the invention, the modifiedmicrobial rhodopsin comprises a mutated proton acceptor proximal to theSchiff Base.

In some or any embodiment or aspect of the invention, the nucleic acidencoding a microbial rhodopsin protein is operably linked to a membranetargeting nucleic acid sequence.

In some or any embodiment or aspect of the invention, the membranetargeting nucleic acid sequence is a plasma membrane targeting nucleicacid sequence.

In some or any embodiment or aspect of the invention, the membranetargeting nucleic acid sequence is a subcellular membrane targetingnucleic acid sequence.

In some or any embodiment or aspect of the invention, the subcellularmembrane is a mitochondrial membrane, an endoplasmic reticulum, asarcoplastic reticulum, a synaptic vesicle, an endosome or a phagosome.

In some or any embodiment or aspect of the invention, the nucleic acidencoding the microbial rhodopsin protein is operably linked to acell-type specific promoter.

In some or any embodiment or aspect of the invention, the nucleic acidencoding the microbial rhodopsin protein is operably linked to anadditional nucleic acid encoding at least one additional fluorescentprotein or a chromophore.

In some or any embodiment or aspect of the invention, the at least oneadditional fluorescent protein is a green fluorescent protein or ahomolog thereof.

In some or any embodiment or aspect of the invention, the at least oneadditional fluorescent protein is capable of indicating ionconcentration in the cell.

In some or any embodiment or aspect of the invention, the at least oneadditional fluorescent protein is capable of indicating ionconcentration in the cell is a calcium indicator.

In some or any embodiment or aspect of the invention, the at least oneadditional fluorescent protein is capable of indicating ionconcentration in the cell is a pH indicator.

In some or any embodiment or aspect of the invention, the cell is adifferentiated cell.

In some or any embodiment or aspect of the invention, the differentiatedcell is a neuron.

In some or any embodiment or aspect of the invention, the differentiatedcell is a cardiomyocyte.

In some or any embodiment or aspect of the invention, the cell is apluripotent cell, a stem cell or an induced pluripotent stem cell.

In some or any embodiment or aspect of the invention, the method furthercomprises a step of differentiating the pluripotent cell, the stem cellor the induced pluripotent stem cell into a differentiated cell.

The method of any one of the claims 80-97, wherein the transducing isperformed using a transient transfection.

In some or any embodiment or aspect of the invention, the transducing isperformed using a stable transfection.

In some or any embodiment or aspect of the invention, the cell istransduced with the isolated and purified nucleic acid as describedherein.

In some embodiments, the method is performed to a non-human cells.

In some embodiments and aspects, the invention provides a cell that hasbeen genetically engineered to express a microbial rhodopsin that is notnaturally present in the cell, and methods of using such cells.

Also provided herein are methods for making an optical sensorcomprising: (a) modifying a microbial rhodopsin protein to reduce theion pumping activity compared to a natural microbial rhodopsin proteinfrom which it is derived, and (b) introducing the modified microbialrhodopsin protein into a cell, thereby producing a genetically encodedoptical sensor. In one embodiment, the step of introducing comprisesintroducing a gene for the modified microbial rhodopsin protein into thecell.

In another aspect, the invention provides for a method for measuring achange in a membrane potential of a cell, the method comprising:measuring an optical signal in a cell, wherein the cell comprises anatural microbial rhodopsin or a modified microbial rhodopsin proteinwith reduced ion pumping activity compared to a natural microbialrhodopsin protein from which it is derived, wherein the microbialrhodopsin or the modified microbial rhodopsin protein localizes to amembrane of the cell and, wherein a change in the level of fluorescenceof the rhodopsin or the modified microbial rhodopsin is indicative of achange in the membrane potential of the cell.

Also described herein is a method of screening for an agent thatmodulates membrane potential, either directly or through an effect onion channel activity, the method comprising: (a) contacting a cell witha candidate agent, wherein the cell comprises a microbial rhodopsin or amodified microbial rhodopsin protein with reduced ion pumping activitycompared to a natural microbial rhodopsin protein from which it isderived, wherein the microbial rhodopsin or the modified microbialrhodopsin protein localizes to a membrane of the cell, and (b) measuringthe optical signal, wherein a change in the level of fluorescence of therhodopsin or the modified microbial rhodopsin in the presence of thecandidate agent is indicative of a change in the membrane potentialacross the membrane of the cell, thereby indicating that the candidateagent modulates membrane potential directly or through an effect on ionchannel activity.

In addition, provided herein is a method of screening for a modulator ofantibiotic efflux in a bacterial cell, the method comprising: (a)contacting a bacterial cell with a candidate agent, wherein the cellcomprises a microbial rhodopsin or a modified microbial rhodopsinprotein having reduced ion pumping activity compared to a naturalmicrobial rhodopsin protein from which it is derived, wherein themicrobial rhodopsin or the modified microbial rhodopsin proteinlocalizes to a membrane of the cell, and (b) measuring thetime-dependent fluctuations in the level of fluorescence of themicrobial rhodopsin or the modified microbial rhodopsin, wherein achange in the fluctuations in the presence of the candidate agent isindicative of a change in fluctuations in the membrane potential acrossthe membrane of the cell, thereby indicating that the candidate agent isa modulator of antibiotic efflux in the bacterial cell. In oneembodiment, the candidate agent is an inhibitor of antibiotic efflux. Inan alternate embodiment, the candidate agent enhances antibiotic efflux.

Also described herein are nucleic acid constructs for expressing amicrobial rhodopsin and a modified microbial rhodopsin protein with areduced ion pumping activity compared to the natural microbial rhodopsinprotein from which it is derived comprising at least one nucleic acidsequence encoding the modified microbial rhodopsin protein that ismodified for codon usage appropriate for the eukaryotic cell.

In another embodiment, described herein are vectors comprising at leastone nucleic acid encoding a voltage indicator protein operably linked toa promoter, e.g., a tissue-specific promoter, such as a neuron-specificpromoter or a cardiac tissue, such as cardiomyocyte-specific promoter.

In some embodiments, the vector also comprises a membrane-targetingsignal sequence to membranes such as the plasma membrane, mitochondria,the endoplasmic reticulum, the sarcoplasmic reticulum, synapticvesicles, and phagosomes. In some embodiments, the vector comprises atleast two nucleic acids each encoding a different voltage indicatorprotein. In some embodiments, the vector further comprises a nucleicacids encoding, e.g., a pH indicator (e.g., pHluorin) or a calciumindicator (e.g., GCaMP3). Use of such a combination enables simultaneousmonitoring of voltage and ion concentration (pH or Ca²⁺, respectively).

In another embodiment, the invention provides a cell expressing, eitherstably or transiently, at least one nucleic acid construct or vectorencoding a microbial rhodopsin or a modified microbial rhodopsindescribed in the specification.

In another aspect, methods are provided for measuring a change in themembrane potential of at least one cell in a cellular circuit, themethod comprising: measuring the level of fluorescence of the microbialrhodopsin or the modified microbial rhodopsin in at least one cell in acellular circuit, wherein the at least one cell in a cellular circuitcomprises a microbial rhodopsin or a modified microbial rhodopsinprotein having reduced ion pumping activity compared to a naturalmicrobial rhodopsin protein from which it is derived, wherein themicrobial rhodopsin or the modified microbial rhodopsin proteinlocalizes to a membrane of the cell, and wherein a change in the levelof fluorescence is indicative of a change in the membrane potentialacross a membrane of the at least one cell in said cellular circuit. Insome and in all aspects of the invention, the microbial rhodopsintargets the plasma membrane of the cell.

Another aspect provided herein is a high-throughput system for opticallymeasuring membrane potential, the system comprising: (a) a plurality ofengineered cells comprising on their membrane at least one modifiedmicrobial rhodopsin protein having reduced ion pumping activity comparedto a natural microbial rhodopsin protein from which it is derived, and,(b) means for detecting fluorescence of the modified microbial rhodopsinprotein.

In one embodiment of the cells or methods described above, the cell is aeukaryotic cell or a prokaryotic cell.

In one embodiment, the eukaryotic cell is a mammalian cell.

In another embodiment of the cell is a stem cell or a pluripotent cell,including an induced pluripotent cell or a stem cell.

In some embodiments, the cell is a progenitor cell, such as a neuralprogenitor or a cardiac progenitor cell.

In some embodiments, the cell is a differentiated cell, such as a neuralcell or a cardiomyocyte.

In another embodiment of the cells or methods described above, theprokaryotic cell is a bacterial cell.

In one embodiment, the step of introducing comprises introducing a genefor the microbial rhodopsin or the modified rhodopsin protein into thecell.

In one and all aspects of the embodiments described herein, the cell isa eukaryotic cell. In one and all embodiments described herein, the celldoes not naturally express a microbial rhodopsin.

In another embodiment of the cells or methods described above, theintroducing is performed using a viral vector.

In another embodiment of the cells or methods described above, theintroducing is performed using electroporation.

In other embodiments of the cells or methods described herein, theintroducing is performed using lipofection, CaPO₄, lipid-mediateddelivery (e.g., liposome, micelle etc.), transfection or transformation.

In another embodiment of the cells or methods described above, theintroducing is performed in vivo into at least one cell in a subject.

In another embodiment of the cells or methods described above, the cellis genetically engineered to encode the modified microbial rhodopsinprotein.

In another embodiment of the cells or methods described above, the cellis a stem cell.

In another embodiment of the cells or methods described above, the stemcell selected from the group consisting of an induced pluripotent cell,an embryonic stem cell, an adult stem cell, and a neuronal stem cell.

In another embodiment of the cells or methods described above, themodified microbial rhodopsin protein further comprises a moiety thatproduces an optical signal.

In another embodiment of the cells or methods described above, themodified microbial rhodopsin protein comprises an amino acid mutationcompared to the natural microbial rhodopsin protein from which it isderived.

In some embodiments, the microbial rhodopsin is a proteorhodopsin.

In some embodiments, the microbial rhodopsin is Archaerhodopsin. In someembodiments, the Archaerhodopsin is Archaerhodopsin 3 (Arch3) or any ofits homologues.

In another embodiment of the cells or methods described above, themodified microbial rhodopsin protein comprising a mutation isgreen-absorbing proteorhodopsin.

In another embodiment of the cells or methods described above, themutation is selected from the group consisting of: D97N, E108Q, E142Q,and L217D.

In another embodiment of the cells or methods described above, themodified microbial rhodopsin protein comprising a mutation isblue-absorbing proteorhodopsin.

In another embodiment of the cells or methods described above, themutation is D85N in bacteriorhodopsin or D99N in blue-absorbingproteorhodopsin.

In another embodiment of the cells or methods described above, the aminoacid mutation is at residue V48, P49, Y57, L92, W86, 1119, M145, W182,Y199, D212, and V213 in bacteriorhodopsin (e.g., a protein encoded bythe nucleic acid sequence GenBank NC_010364.1, sequences1082241-1083029, wherein the nucleotides 1082241 is designated herein asnucleic acid residue 1 or GenBank accession sequence M11720.1) or thehomologous mutations in another microbial rhodopsin.

In one embodiment, the microbial rhodopsin is archaerhodopsin, such asArh-3 (see e.g., Chow, B. Y. et al., Nature 463:98-102 (2010), which isherein incorporated by reference in its entirety). In anotherembodiment, the archaerhodopsin Arh-3 comprises a D95N amino acidmutation or a mutation in a homologous location of anotherarchaerhodopsin. Other achaerhodopsins include, but are not limited toarchaerhodopsin-1 and -2 found in Halorubrum sp. (see, e.g., Enami etal. J Mol Biol. 2006 May 5; 358(3):675-85. Epub 2006 Mar. 3)

In another embodiment of the cells or methods described above, themodified microbial rhodopsin protein is localized to a subcellularcompartment.

In another embodiment of the cells or methods described above, themodified microbial rhodopsin protein is localized to the plasmamembrane, the mitochondrial inner membrane, or the mitochondrial outermembrane.

In another embodiment of the cells or methods described above, themodified microbial rhodopsin protein further comprises a targetingsequence.

In another embodiment of the cells or methods described above, themodified microbial rhodopsin protein comprises a Golgi export sequence,a membrane localization sequence, and/or an endoplasmic reticulum exportsequence.

In another embodiment of the cells or methods described above, themembrane localization sequence comprises a C-terminal signaling sequencefrom the β2 nicotinic acetylcholine receptor.

In another embodiment of the cells or methods described above, themodified microbial rhodopsin protein further comprises a fluorescentmoiety or a chromophore.

In another embodiment of the cells or methods described above, thefluorescence or optical signal is detected by fluorescence, spectralshift fluorescence resonance energy transfer (FRET), rhodopsin opticallock-in imaging (ROLI), Raman, or second harmonic generation (SHG).

In another embodiment of the cells or methods described above, theeukaryotic cell is a neuronal cell.

In another embodiment of the cells or methods described above, themodified microbial rhodopsin protein is expressed in the cell.

In another embodiment of the cells or methods described above, themodified microbial rhodopsin protein is expressed from a nucleic acidsequence encoding the modified microbial rhodopsin protein having amodified codon usage such that the codon usage is appropriate for theeukaryotic cell but the amino acid sequence remains substantiallysimilar to the modified microbial rhodopsin protein.

In another embodiment of the cells or methods described above, thebacterial cell is an antibiotic insensitive strain of bacteria.

In another embodiment of the cells or methods described above, therhodopsin protein further comprises a moiety that produces an opticalsignal.

In another embodiment of the cells or methods described above, themoiety that produces an optical signal comprises a fluorescent moiety ora chromophore.

In another embodiment of the cells or methods described above, theconstruct further comprises a eukaryotic promoter.

In another embodiment of the cells or methods described above, theeukaryotic promoter is an inducible promoter, or a tissue-specificpromoter.

In another embodiment of the cells or methods described above, thecellular circuit comprises at least two neuronal cells.

In another embodiment of the cells or methods described above, the ionpumping activity of the modified microbial rhodopsin protein isinhibited.

In another embodiment of the cells or methods described above, themodified microbial rhodopsin has no measurable ion pumping activity.

In another embodiment of the cells or methods described above, thecandidate agent inhibits, increases, or reduces the ion channelactivity.

EXAMPLES

Voltage is used by biological systems to convey information on bothcellular and organismal scales. Traditional voltage probes rely onelectrodes to make physical contact with the cell and can be used inlimited numbers. Described herein is a new class of fluorescent proteinsbased on microbial rhodopsins, whose fluorescence is exquisitelysensitive to the membrane potential of a cell. The probes, have a farred fluorescent excitiation, a near infrared emission, a millisecondresponse time, and are extremely photostable.

Example 1

Proteorhodopsin as a Fluorescent Sensor of Protons

Proteorhodopsins are a large family of photoactive transmembraneproteins recently discovered in marine bacteria. Green-absorbingproteorhodopsin (GPR) is a light-driven proton pump that converts solarenergy into a proton-motive force used by its host to power cellularmachinery. The directional motion of a proton through GPR is accompaniedby a series of dramatic color shifts in the protein. The approach of thestudy was to determine whether GPR could be run backward: could atransmembrane potential drive a proton through the protein and therebyalter its color? Described herein is a protein-based colorimetricindicator of membrane potential using such an approach.

A covalently bound retinylidene is the visible chromophore of GPR. ASchiff base (SB) links the retinal to the ε-amino group of lysine 231.Early in the wild-type photocycle, a proton leaves the SB, causing ashift in the peak absorption from λ_(max)=535 nm to λ_(max)=421 nm. Anincrease in pH also induces this color shift and indicates that the SBhas a pK_(a)>12. It was reasoned that a change in membrane potentialwould alter the electrochemical potential of the proton on the SB, andthereby affect the pK_(a). The Nernst equation indicates that a ΔV of 59mV at the SB corresponds to a ΔpK_(a) of 1 pH unit. If the pK_(a) becamelower than the ambient pH, the SB loses its proton and a color changewould ensue. Electrochromic responses were shown to occur in bulk in theclosely related protein bacteriorhodopsin when a point mutation was madeto the SB counterion Asp85.

A mutation of the GPR counterion, Asp97Asn, was constructed. The mutanthas been reported to not pump protons in response to light, and has a SBwith pK_(a)=9.9 which is much closer to physiological pH. The purifiedprotein undergoes a visible color change between pH 8.4 and 10.4.Although the pK_(a) was still >2 pH units above ambient, the protein wastested as a voltage sensor. This engineered protein is referred toherein as a Proteorhodopsin Optical Proton Sensor (PROPS).

Optical absorption is insufficiently sensitive to detect color changesin the small amount of PROPS in a single cell. Therefore, a differentspectroscopic readout was sought. Surprisingly, PROPS expressed in E.coli showed clearly visible fluorescence (λ_(exc)=633 nm, λ_(em)=700-750nm) localized to the membrane. Furthermore the fluorescence of purifiedPROPS vanished at high pH, indicating that only the protonated SB isfluorescent with a 633 nm excitation. E. coli−PROPS/+retinal, or+PROPS/−retinal exhibited 100-fold lower fluorescence than cells+PROPS/+retinal (SOM) confirming the emission is arising from thefunctional protein.

PROPS has several photophysical properties that are advantageous forimaging. The red excitation and near infrared emission fall in spectralbands of little background autofluorescence. At pH 7.4, purified PROPShas fluorescence quantum yield (QY)=1.0×10⁻³, while WT GPR hasQY=1.3×10³. The low QY of PROPS is partially offset by its remarkablephotostability. At a laser intensity of 350 mW/cm², PROPS photobleachesto 50% of its initial intensity in >30 minutes, while under the samemeasurement conditions the organic fluorophore Alexa 647 photobleachesto 50% in 24 s. PROPS constitutes a new class of far-red fluorescentprotein with no homology to GFP.

PROPS is Sensitive to the Local Concentration of Protons

To test the sensitivity of PROPS to protons, the protein wassimultaneously imaged with the pH sensitive dye, BCECF, inside intact E.coli treated with CCCP to equalize internal and external pH. A pHtitration of the fluorescence of both fluorophores shows that PROPS isindeed sensitive to protons with the fluorescence decreasing atincreasing pH.

To test the response of PROPS to voltage, E. coli membranes wererendered permeable to K⁺ by treatment with EDTA and valinomycin andsubjected to shocks of KCl using a homebuilt flow chamber. Intensitydifferences were calculated as a function of the imposed membranevoltage. PROPS shows a clear decrease in fluorescence upon a KClup-shock consistent with the model that higher membrane voltage lowersthe fluorescence from the protonated state. Using these measurements, aΔF/F per 100 mV of 500% was calculated. These experiments confirm thatPROPS is sensitive to a linear combination of the internal pH andvoltage.

E. coli Expressing PROPS have Periodic Flashes of Fluorescence

PROPS was expressed in E. coli, the cells were immobilized on a glasscoverslip, and imaged in an inverted epifluorescence microscope whilegently flowing minimal medium at pH 7 over the cells. Many cellsexhibited cell-wide flashes in fluorescence in which the fluorescenceincreased by a factor of up to 8. The flashes occurred simultaneously(to within the 10 ms imaging resolution) and homogeneously over theextent of a cell. Flashes were uncorrelated between neighboring cells.

Within a nominally homogeneous population of cells in a singlemicroscope field of view, a variety of temporal dynamics were observed.The most common flashes had a rise time of 350 ms, a duration of 1 s anda return to baseline over 1000 ms. Many cells flashed periodically, witha typical frequency of 0.2 Hz. Some cells occasionally had “slowflashes”, lasting from 20 s to 40 s. The fast and slow flashes reachedthe same maximum intensity. A third motif was a “ringing pattern” inwhich the fluorescence oscillations became smaller in amplitude andhigher in frequency until the cell settled at an intermediate intensity.Some cells were quiescent for many minutes, flashed once, and thenreturned to darkness; others had periods of quiescence punctuated bybrief bursts of flashing. Some cells were permanently bright, and somewere permanently dark. Four strains of E. coli showed blinks with twodifferent plasmids encoding PROPS (arabinose or IPTG induction).Flashing occurred in cells immobilized via poly-L-lysine, cells left tosettle on an uncoated coverslip, and cells immobilized on an agarosepad. Flashing was observed on two independent microscopic imagingsystems.

It was sought to determine whether flashing was induced by theexpression of PROPS and/or by the laser used in imaging. The same fieldof cells was monitored under increasing laser power over 2 orders ofmagnitude. The blinking was unchanged until a threshold intensity of 100W/cm² at which point the blinking rose dramatically. The cause of theincreased blinking is unclear. The sharp increase in blinking allows tworegimes of PROPS (i) monitoring endogenous activity at low powers (<50W/cm²) and (ii) enhancing activity at high powers (>100 W/cm²). Toincrease the fraction of cells undergoing blinking, the rest of thestudies were conducted in the high power regime.

It was also observed that strain JY29 showed some “sub-threshold”flashes with rise and fall times as brief as 4 ms. This observation issignificant because it establishes that PROPS responds to voltagefluctuations on a timescale comparable to the duration of a neuronalaction potential (1 ms). At present it is not clear whether the observed4 ms timescale is set by the response speed of PROPS or by the intrinsicvoltage dynamics in E. coli.

Flashes are Due to Swings in Voltage, not pH

PROPS are sensitive to both voltage and pH, but the transient blinks influorescence could be a function of the internal pH (pH_(i)), theexternal pH (pH_(o)), and the membrane potential (ΔV). The study soughtto determine this unknown response function, and furthermore todetermine whether flashing was caused by changes in pH_(i), ΔV. or both.Independent measures of these quantities were deemed necessary.

To measure pH_(i), cells were incubated with EDTA and a fluorescentindicator of pH, BCECF-AM. This dye is taken up by the cells andconverted to a fluorescent membrane-impermeable form. The fluorescenceof the BCECF and of the PROPS was simultaneously measured. The BCECFfluorescence was largely constant as pH_(o) was varied from 6.5-9,consistent with earlier findings that E. coli maintain homeostasis ofpH_(i)˜7.8 over this range of pH_(o). The PROPS baseline (non-blinking)fluorescence was also largely constant over this range of pH_(o),indicating that the SB in PROPS is not exposed to the extracellularmedium. The ionophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP)was then added, which renders the cell membrane permeable to protons sothat changes in pH_(o) induce corresponding changes in pH_(i). Both theBCECF and the PROPS fluorescence changed much more in response tochanged pH_(o), indicating that PROPS is sensitive to pH_(i). CCCPtreated cells with BCECF responded to step changes in pH_(o) fast enoughthat if there were a change in pH, during a flash, a change in BCECFfluorescence would be detectable.

A fresh sample of blinking cells was prepared with EDTA and BCECF andtwo-color movies were recorded showing simultaneous fluorescence ofBCECF and PROPS (data not shown). These cells continued to flash in thePROPS channel, but the BCECF fluorescence remained constant to withinmeasurement error. This indicates that there is little or no change inpH, during a blink.

To correlate flashing with ΔV, PROPS fluorescence and flagellar rotationof E. coli strain JY29 were simultaneously observed. These cells have asticky flagellum which binds to a glass coverslip, causing the cell bodyto rotate at an angular velocity proportional to the cellular PMF.Strain JY29 lacks CheY and thus its flagellar motor does not reversedirection. Flashes were associated with slowing or pauses of therotation indicating that blinks occur as a lowering of the cellular PMF.Since pH_(o) and pH_(i) remain constant, the membrane potential must bereduced to account for the change in PMF.

Blinks are Sensitive to pH, Benzoate, and [Na⁺] and [K⁺].

Flashing cells were subjected to chemical perturbations in an effort todeduce the mechanism of the voltage changes. In unstirred medium in asealed chamber, the flashing ceased over ˜15 minutes; but re-startedupon addition of freshly oxygenated medium. Removal of oxygen from themedium using Oxyrase also reversibly eliminated flashing. From theseexperiments it was concluded that flashing requires aerobic respiration.Under continuous gentle flow of aerated minimal medium, cells flashedcontinuously for >1 hr. Cells stored in minimal media for several daysat 4° C. started flashing when warmed to room temperature.

Flashing was highly sensitive to pH_(o), occurring only between pH 7 and9. Flashing frequencies increased and on-time decreased at lower pH.Flashing was more likely to be periodic at lower pH. Extremes of pH (<6or >9.5) irreversibly eliminated flashing, causing cells to enter thebright state in an abrupt transition. Addition of CCCP (20 μg/mL)rapidly and irreversibly eliminated the blinking, causing all cells tobecome bright.

Ionic shocks were tested to determine the ions conducted during thevoltage swing. Cells in buffer containing just Na⁺, K⁺, Cl⁻, and PO₄were able to blink for >30 minutes at pH 7.5. Removal of just Na⁺ or K⁺did not immediately stop blinking, but removal of both ions causeddramatic fluorescence changes. Likewise, a 2 M KCl shock induceddramatic effects, but a 2 M NaCl shock did not. The evidence does notpoint to specific ions, but rather a channel that is capable oftransporting multiple types of ions. Further tests on the mechanisms ofblinking and efflux are being conducted with genetic knockouts.

The development of PROPS represents an important step in molecularprobes. With the advent of the optogenetic tools for controlling neuralactivity based on channel rhodopsin II and halorhodopsin, microbialrhodopsins have gained interest as tools for neuroscience. PROPS adds animportant tool in neuroscience through its ability to probe electricalactivity with light. The far red shifted excitation and emission spectraof PROPS can combine easily with current efforts of channel rhodopsin toprovide fully optical control and read-out of membrane potentials.

Using the unique capabilities of PROPS spontaneous electrical activityin respirating E. coli was discovered. The discrete nature of the spikesis reminiscent of localization of the Crz1 protein to the nucleus ofyeast during Ca⁺⁺ adaption, although the localization on yeast occurs ona much slower timescale (minutes rather than seconds.) The biologicalsignificance of electrical spiking in E. coli is at present unclear.

PROPS can also be used to determine (i) the molecular components andmechanisms behind bacterial blinking, (ii) the biological role that eachof the three types of blinks serve, (iii) other cellular behaviors, and(iv) blinking patterns of other bacterial strains. In one embodiment,GPR is engineered to have a higher fluorescent quantum yield. GPR, itsmutants, and other microbial rhodopsins are a promising family offluorescent indicators of membrane potential. In one embodiment, PROPSand its variants may be used to monitor changes in membrane potential inother types of electrically active cells, such as neurons.

Example 2 Arch 3 can be Run Backward and Thereby Provide a VoltageSensor

Archaerhodopsin 3 (Arch 3) from Halorubrum sodomense is a light-drivenoutward proton pump, capturing solar energy for its host (Ihara, K. etal. Evolution of the archaeal rhodopsins: evolution rate changes by geneduplication and functional differentiation. J. Mol. Biol. 285, 163-174(1999)). Recently Arch 3 was expressed in mammalian neurons, wherein itenabled optical silencing of neural activity, and was shown to beminimally perturbative to endogenous function in the dark (Chow, B. Y.et al. High-performance genetically targetable optical neural silencingby light-driven proton pumps. Nature 463, 98-102 (2010)). We have nowdemonstrated that Arch 3 can be run backward: that a membrane potentialcan alter the optical properties of the protein, and thereby provide avoltage sensor that function through a mechanism similar to PROPS.

At neutral pH Arch 3 was pink, but at high pH the protein turned yellow(FIG. 8A), with a pKa for the transition of 10.1. Based on homology toother microbial rhodopsins (Lanyi, J. K. Bacteriorhodopsin. Annu. Rev.Physiol. 66, 665-688 (2004); Friedrich, T. et al. Proteorhodopsin is alight-driven proton pump with variable vectoriality. J. Mol. Biol. 321,821-838 (2002)), we attributed the pH-induced color change todeprotonation of the Schiff Base (SB) which links the retinalchromophore to the protein core (Dioumaev, A. K. et al. Proton transfersin the photochemical reaction cycle of proteorhodopsin. Biochemistry 41,5348-5358 (2002)). We reasoned that a change in membrane potential mightchange the local electrochemical potential of the proton at the SB,tipping the acid-base equilibrium and inducing a similar color shift(FIG. 6A). This mechanism of voltage-induced color shift has previouslybeen reported in dried films of bacteriorhodopsin (Kolodner, P.,Lukashev, E. P., Ching, Y. & Rousseau, D. L. Electric-field-inducedSchiff-base deprotonation in D85N mutant bacteriorhodopsin. Proc. Nat.Acad. Sci. U.S.A 93, 11618-11621 (1996)), and formed the basis ofvoltage sensitivity in PROPS (Kralj, J. M., Hochbaum, D. R., Douglass,A. D. & Cohen, A. E. Electrical spiking in Escherichia coli probed witha fluorescent voltage indicating protein. Science 333, 345-348 (2011)).

Changes in optical absorption would be challenging to detect in a singlecell, due to the small quantity of protein available. However, mostmicrobial rhodopsins are weakly fluorescent (Lenz, M. O. et al. Firststeps of retinal photoisomerization in proteorhodopsin. Biophys. J. 91,255-262 (2006)), so we characterized purified Arch 3 as a prospectivefluorescent indicator (Table 6). At neutral pH, Arch emitted far redfluorescence (λem=687 nm), while at high pH Arch was not fluorescent(FIG. 6B, FIG. 17). The fluorescence quantum yield of Arch was low(9×10−4) but the photostability was comparable to members of the GFPfamily (Shaner, N. C., Steinbach, P. A. & Tsien, R. Y. A guide tochoosing fluorescent proteins. Nat. Meth. 2, 905 (2005)), yieldingapproximately 25% as many photons prior to photobleaching as eGFP. Thebroad absorption peak enabled excitation at λ=640 nm, a wavelength wherefew other cellular components absorb, and the far red emission occurredin a spectral region of little background autofluorescence.

Fluorescence of Arch 3 in HEK 293 cells was readily imaged in aninverted fluorescence microscope with red illumination (λ=640 nm, I=540W/cm²), a high numerical aperture objective, a Cy5 filter set, and anEMCCD camera. The cells exhibited fluorescence predominantly localizedto the plasma membrane (FIG. 6C). Cells not expressing Arch were notfluorescent. Cells showed 17% photobleaching over a continuous 10-minuteexposure, and retained normal morphology during this interval.

The fluorescence of HEK cells expressing Arch was highly sensitive tomembrane potential, as determined via whole-cell voltage clamp. Wedeveloped an algorithm to combine pixel intensities in a weighted sumsuch that the output, was a nearly optimal estimate of membranepotential V determined by conventional electrophysiology. FIG. 6C showsan example of a pixel-weight matrix, indicating that thevoltage-sensitive protein was localized to the cell membrane;intracellular Arch contributed fluorescence but no voltage-dependentsignal. The fluorescence increased by a factor of 2 between −150 mV and+150 mV, with a nearly linear response throughout this range (FIG. 6D).The response of fluorescence to a step in membrane potential occurredwithin the 500 μs time resolution of our imaging system on both therising and falling edge (FIG. 6E). Application of a sinusoidally varyingmembrane potential led to sinusoidally varying fluorescence; at f=1 kHz,the fluorescence oscillations retained 55% of their low-frequencyamplitude (FIG. 18). Arch reported voltage steps as small as 10 mV, withan accuracy of 625 μV/(Hz)^(1/2) over timescales <12 s (FIG. 19). Overlonger timescales laser power fluctuations and cell motion degraded theaccuracy.

We tested Arch 3 as a voltage indicator in cultured rat hippocampalneurons, using viral delivery. Neurons expressing Arch showedvoltage-dependent changes in fluorescence localized to the cellmembrane. Under whole cell current clamp, cells exhibited spiking uponinjection of current pulses of 200 pA. Individual spikes wereaccompanied by clearly identifiable increases of fluorescence (FIG. 7A).At a 2 kHz image acquisition rate, the signal-to-noise ratio in thefluorescence (spike amplitude:baseline noise) was 10.5. A spike-findingalgorithm correctly identified 99.6% of the spikes (based on comparisonto simultaneously recorded membrane potential), with a false-positiverate of 0.7% (n=269 spikes) (FIG. 7B). Single cells were observed for upto 4 minutes of cumulative exposure, with no detectable change inresting potential or spike frequency.

We imaged the dynamics of action potentials with sub-cellular resolutionby averaging multiple temporally registered movies of single spikes(FIG. 7C). In and near the soma, the optically determined waveform ofthe action potential was uniform and matched the electrically recordedwaveform. However in very thin processes the peak of the actionpotential lagged by up to 1 ms (FIG. 7D). These observations areconsistent with multiple-patch recordings on single neurons (Stuart, G.J. & Sakmann, B Active propagation of somatic action potentials intoneocortical pyramidal cell dendrites. Nature 367, 69-72 (1994)); butsuch recordings are technically demanding and only probe the variationin membrane potential at a small number of points. The present resultssuggest that Arch may be used to map intracellular dynamics of actionpotentials.

In the absence of added retinal, neurons expressing Arch showed clearlyidentifiable fluorescence flashes accompanying individual spikes (FIG.20), indicating that neurons contained sufficient endogenous retinal topopulate some of the protein. Experiments with Arch and other microbialrhodopsins in vivo have shown that endogenous retinal is sufficient foroptogenetic control of neural activity 25. Thus Arch may function as avoltage indicator in vivo without exogenous retinal.

Illumination at 640 nm was far from the peak of the Arch absorptionspectrum (λ=558 nm), but the imaging laser nonetheless inducedphotocurrents of 10-20 pA in HEK cells expressing Arch 3 (FIG. 8A). Wesought to develop a mutant which did not perturb the membrane potential,yet which maintained voltage sensitivity. The mutation D85N inbacteriorhodopsin eliminated proton pumping 26, so we introduced thehomologous mutation, D95N, into Arch 3. This mutation eliminated thephotocurrent (FIG. 8A) and shifted several other photophysicalproperties of importance to voltage sensing (Table 6, FIG. 8, FIG. 21).Movies of the fluorescence response to changes in membrane potentialwere also taken and showed the result visually. Arch D95N was moresensitive and brighter than Arch 3 WT, but had a slower response (FIG.8A-8C).

Under illumination conditions typically used for imaging neural activity(I=1800 W/cm2 in total internal reflection (TIR) mode), thelight-induced outward photocurrent was typically 10 pA in neuronsexpressing Arch 3 WT. Under current-clamp conditions this photocurrentshifted the resting potential of the neurons by up to −20 mV. Forneurons near their activation threshold, this photocurrent couldsuppress firing (FIG. 9A), so we explored the non-pumping variant D95Nas a voltage indicator in neurons. Illumination of Arch D95N did notperturb membrane potential in neurons (FIG. 9B).

Arch 3 D95N reported neuronal action potentials on a single-trial basis(FIG. 9C). The response to a depolarizing current pulse was dominated bythe slow component of the step response; yet the fast component of theresponse was sufficient to indicate action potentials.

Protein Purification

A lentiviral backbone plasmid encoding Arch 3-EGFP (FCK:Arch 3-EGFP) wasa generous gift from Dr. Edward Boyden (MIT). The gene for the rhodopsinwas cloned into pet28b vector using the restriction sites EcoRI andNcoI. The D95N mutation was created using the QuikChangell kit (Agilent)using the forward primer (5′-TTATGCCAGGTACGCCAACTGGCTGTTTACCAC) (SEQ IDNO: 22) and the reverse primer (5′-GTGGTAAACAGCCAGTTGGCGTACCTGGCATAA)(SEQ ID NO: 23).

Arch and its D95N mutant were expressed and purified from E. coli,following Bergo, V., et al. (Conformational changes detected in asensory rhodopsin II-transducer complex. J. Biol. Chem. 278, 36556-36562(2003)). Briefly, E. coli (strain BL21, pet28b plasmid) was grown in 1 Lof LB with 100 μg/mL kanamycin, to an O.D 600 of 0.4 at 37° C. All-transretinal (5 μM) and inducer (IPTG 0.5 mM) were added and cells were grownfor an additional 3.5 hours in the dark. Cells were harvested bycentrifugation and resuspended in 50 mM Tris, 2 mM MgCl₂ at pH 7.3 andlysed with a tip sonicator for 5 minutes. The lysate was centrifuged andthe pellet was resuspended in PBS supplemented with 1.5% dodecylmaltoside (DM). The mixture was homogenized with a glass/teflon PotterElvehjem homogenizer and centrifuged again. The supernatant wasconcentrated and washed using a centricon 30k MWCO filter to a finalvolume of 3 mL.

Spectroscopic Characterization of Arch WT and D95N

The absorption spectra of purified Arch 3 WT and D95N were determinedusing an Ocean Optics USB4000 spectrometer with a DT-MINI-2-GS lightsource. Absorption spectra, for Arch 3 WT and D95N, were measured as afunction of pH between pH 6 and 11. To determine the fluorescenceemission spectra, proteins in a quartz cuvette were illuminated with theuncollimated beam of a 100 mW, 532 nm laser (Dragon Lasers, 532GLM100)or a 25 mW, 633 nm HeNe laser (Spectra-Physics). Scattered laser lightwas blocked with a 532 nm Raman notch filter (Omega Optical, XR03) or a710/100 emission filter (Chroma), and fluorescence was collectedperpendicular to the illumination with a 1000 micron fiber, which passedthe light to an Ocean Optics QE65000 spectrometer. Spectra wereintegrated for 2 seconds.

The fluorescence quantum yields of Arch 3 WT and D95N were determined bycomparing the integrated emission intensity to emission of a sample ofthe dye ALEXA FLUOR® 647 Conjugate. Briefly, the concentrations ofmicromolar solutions of dye and protein were determined using a visibleabsorption spectrum. We used the extinction coefficients of 270,000 M⁻¹cm⁻¹ for ALEXA FLUOR® 647 and 63,000 M⁻¹ cm⁻¹ for Arch 3 WT and D95N,assuming that these microbial rhodopsins have the same extinctioncoefficient as bacteriorhodopsin. The dye solution was then diluted1:1000 to yield a solution with comparable fluorescence emission to theArch 3. The fluorescence emission spectra of dye and protein sampleswere measured with 633 nm excitation. The quantum yield was thendetermined by the formula

${QY}_{Arch} = {\frac{{Fl}_{Arch}}{{Fl}_{Alexa}}*\frac{ɛ_{Alexa}}{ɛ_{Arch}}*\frac{c_{Alexa}}{c_{Arch}}*{QY}_{Alexa}}$

where Fl is the integrated fluorescence from 660 to 760 nm, E is theextinction coefficient at 633 nm and c is the concentration.

Relative Photostability of Arch 3 and eGFP

To perform a direct comparison of photostability of Arch 3 and eGFP westudied the photobleaching of the Arch 3-eGFP fusion. This strategyguaranteed a 1:1 stoichiometry of the two fluorophores, simplifying theanalysis. The experiments were performed on permeabilized cells, in themicroscope, with video recording as the cells photobleached. We firstrecorded a movie of photobleaching of Arch 3 under 640 nm illumination;then on the same field of view we recorded photobleaching of eGFP under488 nm illumination, with illumination intensity adjusted to yieldapproximately the same initial count rate as for Arch 3. Fluorescencebackground levels were obtained from nearby protein-free regions of eachmovie and were subtracted from the intensity of the protein-containingregions. The area under each photobleaching timetrace was calculated,yielding an estimate of the total number of detected photons from eachfluorophore. The eGFP emission (λ_(max)=509 nm) and the Arch emission(λ_(max)=687 nm) were collected through different emission filters, sothe raw counts were corrected for the transmission spectra of thefilters and the wavelength-dependent quantum yield of the EMCCD camera.The result was that the relative number of photons emitted prior tophotobleaching for eGFP:Arch 3 WT was 3.9:1, and for eGFP:Arch D95N thisratio was 10:1.

HEK Cell Culture

HEK-293 cells were grown at 37° C., 5% CO₂, in DMEM supplemented with10% FBS and penicillin-streptomycin. Plasmids were transfected usingLIPOFECTAMINE™ and PLUS reagent (Invitrogen) following themanufacturer's instructions, and assayed between 48-72 hours later. Theday before recording, cells were re-plated onto glass-bottom dishes(MatTek) at a density of ˜5000 cells/cm².

The concentration of endogenous retinal in the HEK cells was not known,so the cells were supplemented with retinal by diluting stock retinalsolutions (40 mM, DMSO) in growth medium to a final concentration of 5μM, and then placing the cells back in the incubator for 1-3 hours. Allimaging and electrophysiology were performed in Tyrode's buffer(containing, in mM: 125 NaCl, 2 KCl, 3 CaCl₂, 1 MgCl₂, 10 HEPES, 30glucose pH 7.3, and adjusted to 305-310 mOsm with sucrose). Only HEKcells having reversal potentials between −10 and −40 mV were included inthe analysis.

Microscopy

Simultaneous fluorescence and whole-cell patch clamp recordings wereacquired on a home-built, inverted epifluorescence microscope at roomtemperature. A diode laser operating at 100 mW, 640 nm (CrystaLaser,DL638-100-0) provided wide-field illumination at an intensity typicallybetween 500-2000 W/cm². To minimize background signal from out-of-focusdebris, illumination was often performed in through-the-objective totalinternal reflection fluorescence (TIRF) mode. Fluorescence emission wascollected using a 60×, 1.45 NA oil immersion objective (Olympus), andseparated from scattered excitation using a 660-760 nm bandpass emissionfilter (Chroma). For fast imaging of dynamic fluorescence responses andaction potentials, images were acquired on an Andor iXon⁺ 860 cameraoperating at up to 2,000 frames/s (using a small region of interest andpixel binning) Slower images with higher spatial resolution wereacquired on an Andor iXon⁺ 897 EMCCD. Custom software written in LabView(National Instruments) was used to synchronize illumination, collectionof images, recording of membrane potential and cell current, andapplication of electrical stimuli to the cell.

Electrophysiology

Filamented glass micropipettes (WPI) were pulled to a tip resistance of3-10 MΩ, fire polished, and filled with internal solution (containing,in mM: 125 Potassium gluconate, 8 NaCl, 0.6 MgCl₂, 0.1 CaCl₂, 1 EGTA, 10HEPES, 4 Mg-ATP, 0.4 Na-GTP, pH 7.3; adjusted to 295 mOsm with sucrose).The micropipettes were positioned with a Burleigh PCS 5000micromanipulator. Whole-cell, voltage clamp recordings were acquiredusing an AxoPatch 200B amplifier (Molecular Devices), filtered at 2 kHzwith the internal Bessel filter, and digitized with a NationalInstruments PCIE-6323 acquisition board at 10 kHz. Ambient 60 Hz noisewas removed using a HumBug Noise Eliminator (AutoMate Scientific). Forexperiments requiring rapid modulation of transmembrane potential,series resistance and whole-cell capacitance were predicted to 95% andcorrected to ˜50%. Electrical stimuli were generated using the PCIE-6323acquisition board and sent to the AXOPATCH™, which then applied thesesignals in either constant current or constant voltage mode.

Measurements of photocurrents were performed on HEK cells held involtage clamp at 0 mV while being exposed to brief (200 ms) pulses ofillumination at 640 nm at an intensity of 1800 W/cm².

All experiments were performed at room temperature.

Estimates of Membrane Potentials from Fluorescence Images

A common practice in characterizing fluorescent voltage indicators is toreport a value of ΔF/F per 100 mV of membrane potential. We feel thatthis parameter is of limited use, for several reasons. First, the valueof ΔF/F is highly sensitive to the method of background subtraction,particularly for indicators in which F approaches zero at some voltage.Second, ΔF/F contains no information about signal-to-noise ratio: oneindicator may have small values of F and of ΔF and another may havelarge values, but the ratio ΔF/F might be the same. Third, the ratioΔF/F contains no information about the temporal stability of thefluorescence. Fluctuations may arise due to intracellular transport,photobleaching, or other photophysics.

We therefore sought a measure of the performance of a voltage indicatorwhich reported the information content of the fluorescence signal. Wesought an algorithm to infer membrane potential from a series offluorescence images. We used the accuracy with which the estimatedmembrane potential matched the true membrane potential (as reported bypatch clamp recording) as a measure of indicator performance.

The estimated membrane potential, {circumflex over (V)}_(FL) (t), wasdetermined from the fluorescence in two steps. First we trained a modelrelating membrane potential to fluorescence at each pixel. We used thehighly simplified model that the fluorescence signal, S_(i)(t), at pixeli and time t, is given by:S _(i)(t)=a _(i) +b _(i) V(t)+ε_(i)(t),  [S1]

where a_(i) and b_(i) are position-dependent but time-independentconstants, the membrane potential V(t) is time-dependent but positionindependent, and ε_(i)(t) is spatially and temporally uncorrelatedGaussian white noise with pixel-dependent variance:

ε_(i)(t ₁)ε_(j)(t ₂)

=σ_(i) ²δ_(i,j)δ(t ₁ −t ₂),

where

indicates an average over time.

This model neglects nonlinearity in the fluorescence response tovoltage, finite response time of the protein to a change in voltage,photobleaching, cell-motion or stage drift, and the fact that ifε_(i)(t) is dominated by shot-noise then its variance should beproportional to S_(i)(t), and its distribution should be Poisson, notGaussian. Despite these simplifications, the model of Eq. S1 providedgood estimates of membrane potential when calibrated from the samedataset to which it was applied.

The pixel-specific parameters in Eq. 1 are determined by a least-squaresprocedure, as follows. We define the deviations from the meanfluorescence and mean voltage byδS _(i)(t)=S _(i)(t)−

S _(i)(t)

δV(t)=V(t)−

V(t)

.

Then the estimate for the slope {circumflex over (b)}_(i) is:

${{\hat{b}}_{i} = \frac{\left\langle {\delta\; S_{i}\delta\; V} \right\rangle}{\left\langle {\delta\; V^{2}} \right\rangle}},$and the offset is:â _(i) =

S _(i)

−{circumflex over (b)} _(i)

V

.

A pixel-by-pixel estimate of the voltage is formed from:

${{\hat{V}}_{i}(t)} = {\frac{S_{i}(t)}{{\hat{b}}_{i}} - {\frac{{\hat{a}}_{i}}{{\hat{b}}_{i}}.}}$

The accuracy of this estimate is measured byζ_(i) ²=

({circumflex over (V)} _(i)(t)−V(t)²

.

A maximum likelihood weight matrix is defined by:

$\begin{matrix}{w_{i} \equiv {\frac{1/ϛ_{i}^{2}}{\sum\limits_{i}^{\;}{1/ϛ_{i}^{2}}}.}} & \lbrack{S2}\rbrack\end{matrix}$

This weight matrix favors pixels whose fluorescence is an accurateestimator of voltage in the training set.

To estimate the membrane potential, the pixel-by-pixel estimates arecombined according to:

$\begin{matrix}{{{\hat{V}}_{FL}(t)} = {\sum\limits_{i}^{\;}{w_{i}{{\hat{V}}_{i}(t)}}}} & \lbrack{S3}\rbrack\end{matrix}$

Within the approximations underlying Eq. S1, Eq. S3 is the maximumlikelihood estimate of V(t).

Ramp and Step-response of Arch 3 WT and D95N

To measure fluorescence as a function of membrane potential, a trianglewave was applied, with amplitude from −150 mV to +150 mV and period 12s, with video recording at 100 ms per frame. A pixel weight matrix wascalculated according to Eq. S2 and applied to the movie images togenerate a fluorescence number for each frame. These fluorescence valueswere divided by their minimum value (at V=−150 mV). The result isplotted as a function of V in FIGS. 7 and 8. This procedurepreferentially weighted data from pixels at the cell membrane, but didnot entail any background subtraction. Comparable results were obtainedby manually selecting pixels corresponding to a region of plasmamembrane, and plotting their intensity as a function of V, withoutbackground subtraction. Background subtraction from the raw fluorescencewould have yielded considerably larger values of ΔF/F.

The step response was measured in a similar manner, except that testwaveforms consisted of a series of voltage pulses, from −70 mV to +30 mVwith duration 300 ms and period 1 s. Cells were subjected to 20repetitions of the waveform, and the fluorescence response was averagedover all iterations.

Frequency-dependent Response Functions of Arch 3 WT and D95N

Test waveforms consisted of a concatenated series of sine waves, each ofduration 2 s, amplitude 100 mV, zero mean, and frequencies uniformlyspaced on a logarithmic scale between 1 Hz and 1 kHz (31 frequenciestotal). The waveforms were discretized at 10 kHz and applied to thecell, while fluorescence movies were acquired at a frame rate of 2 kHz.

The model parameters for extracting {circumflex over (V)}_(FL) (t) werecalculated from the fluorescence response to low frequency voltages.These parameters were then used to calculate an estimated voltage at allfrequencies.

The applied voltage was downsampled to 2 kHz to mimic the response of avoltage indicator with instantaneous response. For each appliedfrequency, the Fourier transform of {circumflex over (V)}_(FL) (t) wascalculated and divided by the Fourier transform of the downsampled V(t).The amplitude of this ratio determined the response sensitivity. It wascrucial to properly compensate pipette resistance and cell membranecapacitance to obtain accurate response spectra. Control experiments oncells expressing membrane-bound GFP showed no voltage-dependentfluorescence.

The power spectrum of {circumflex over (V)}_(FL) (t) under constant V=0was also measured to enable calculations of signal-to-noise ratio forany applied V(t).

Molecular Biology and Virus Production

Plasmids encoding Arch-EGFP (FCK:Arch-EGFP) were either used directlyfor experiments in HEK cells, or first used to produce VSVg-pseudotypedvirus according to published methods (Chow, B. Y. et al.High-performance genetically targetable optical neural silencing bylight-driven proton pumps. Nature 463, 98-102 (2010)). For pseudotyping,HEK-293 cells were co-transfected with pDelta 8.74, VSVg, and either ofthe Arch backbone plasmids using LIPOFECTAMINE and PLUS reagent(Invitrogen). Viral supernatants were collected 48 hours later andfiltered using a 0.45 inn membrane. The virus medium was used to infectneurons without further concentration.

The D95N mutation was introduced using the QUICKCHANGE® kit(Stratagene), according to the manufacturer's instructions using thesame primers as the E. coli plasmid.

Neuronal Cell Culture

E18 rat hippocampi were purchased from BrainBits and mechanicallydissociated in the presence of 1 mg/mL papain (Worthington) beforeplating at 5,000-30,000 cells per dish on poly-L-lysine andMatrigel-coated (BD Biosciences) glass-bottom dishes. Cells wereincubated in N+ medium (100 mL Neurobasal medium, 2 mL B27 supplement,0.5 mM glutamine, 25 μM glutamate, penicillin-streptomycin) for 3 hours.An additional 300 μL virus medium was added to the cells and incubatedovernight, then brought to a final volume of 2 mL N+ medium. After twodays, cells were fed with 1.5 mL N+ medium. Cells were fed with 1 mL N+medium without glutamate at 4 DIV, and fed 1 mL every 3-4 days after.Cells were allowed to grow until 10-14 DIV at which point they were usedfor experiments.

Whole-cell current clamp recordings were obtained from mature neuronsunder the same conditions used for HEK cells recordings. Seriesresistance and pipette capacitance were corrected. Only neurons havingresting potentials between −50 and −70 mV were used in the analysis.

Spike Sorting

A spike identification algorithm was developed that could be appliedeither to electrically recorded V(t) or to optically determined{circumflex over (V)}(t). The input trace was convolved with a referencespike of duration 10 ms. Sections of the convolved waveform that crosseda user-defined threshold were identified as putative spikes. Multiplespikes that fell within 10 ms (a consequence of noise-induced glitchesnear threshold) were clustered and identified as one.

Table 6 shows Optical and electrical response of Arch 3 WT and ArchD95N. ⁽¹⁾ Excitation at λ=532 nm. ⁽²⁾ Absorption spectra calibratedassuming the same peak extinction coefficient as Bacteriorhodopsin,63,000 M⁻¹ cm⁻¹ (Ref. ³²). ⁽³⁾ Determined via comparison to Alexa 647with excitation at λ=633 nm. ⁽⁴⁾ Measured in a 1:1 fusion with eGFP. ⁽⁵⁾Determined via singular value decomposition on absorption spectra. ⁽⁶⁾Determined from step-response. Arch D95N has a minor component of itsresponse (20%) that is fast (<0.5 ms). ⁽⁷⁾ {circumflex over (V)}_(FL) isthe membrane potential estimated from fluorescence. Noise determined atfrequencies f≥0.1 Hz in HEK cells.

TABLE 6 λ_(max) λ_(max) Photostability abs em⁽¹⁾ ε₆₃₃ ⁽²⁾ relative topK_(a) τ_(response) ⁽⁶⁾ Noise in {circumflex over (V)}_(FL) ⁽⁷⁾ Photo-(nm) (nm) (M⁻¹cm⁻¹) QY⁽³⁾ eGFP⁽⁴⁾ of SB⁽⁵⁾ (ms) (μV/Hz^(1/2)) currentArch 558 687 6,300 9 × 10⁻⁴ 0.25 10.1 <0.5 625 yes WT Arch 585 68737,500 4 × 10⁻⁴ 0.1 8.9 41 260 no D95N

The references cited throughout the specification and examples arehereby incorporated by reference in their entirety.

We claim:
 1. A system for measuring membrane potential in a cellcomprising: a cell expressing a nucleic acid encoding a microbialrhodopsin protein fused to an additional fluorescent protein, whereinthe cell is excited with light of at least one wavelength; and amicroscope for detecting at least one optical signal from the cell,wherein the level of fluorescence emitted by the cell compared to areference is indicative of the membrane potential of the cell.
 2. Thesystem of claim 1 further comprising a computer for analyzing the levelof fluorescence emitted by the cell compared to a reference.
 3. Thesystem of claim 1, wherein the microbial rhodopsin protein is a modifiedmicrobial rhodopsin protein.
 4. The system of claim 1, wherein thefluorescence emission of the additional fluorescent protein overlapswith the absorption of the microbial rhodopsin protein.
 5. The system ofclaim 1, wherein the carboxylic amino acid on the third transmembranehelix of the microbial rhodopsin protein comprises a proton acceptorproximal to a Schiff base.
 6. The system of claim 1, wherein themicrobial rhodopsin protein is a member of the proteorhodopsin family ofproteins or a member of the archaerhodopsin family of proteins.
 7. Thesystem of claim 1, wherein the modified microbial rhodopsin protein is:a mutation to a carboxylic amino acid on a third transmembrane helix ofthe microbial rhodopsin protein; and has reduced ion pumping activitycompared to natural microbial rhodopsin protein from which it isderived.
 8. The system of claim 1, wherein the microbial rhodopsinprotein is Proteorhodopsin Optical Proton Sensor, or a geneticallyencoded voltage indicator.
 9. The system of claim 1, wherein the atleast one wavelength is a wavelength between δ=594-645 nm.
 10. Thesystem of claim 1, wherein the cell is selected from prokaryotic cells.11. The system of claim 1, wherein the cell is selected from eukaryoticcells.
 12. The system of claim 1, wherein the cell is selected frommammalian cells.
 13. The system of claim 1, wherein the cell is selectedfrom the group consisting of stem cells, pluripotent cells, progenitorcells, and induced pluripotent cells.
 14. The system of claim 1, whereinthe cell is selected from the group consisting of neurons andcardiomyocytes.
 15. The system of claim 1, wherein the additionalfluorescent protein is a protein that is capable of indicating ionconcentration in the cell.
 16. The system of claim 15, wherein theadditional fluorescent protein that is capable of indicating ionconcentration is a calcium indicator.
 17. The system of claim 15,wherein the additional fluorescent protein that is capable of indicatingion concentration is a pH indicator.
 18. The system of claim 4, whereinthe additional fluorescent protein is capable of undergoing nonradiativefluorescence resonance energy transfer to the microbial rhodopsinprotein, with a rate of energy transfer dependent on membrane potential.19. The system of claim 1, wherein the additional fluorescent protein isselected from the group consisting of a green fluorescent protein,mOrange2, and eGFP.
 20. The system of claim 1, wherein the microbialrhodopsin protein is a modified microbial rhodopsin protein selectedfrom the group consisting of Ar1-D97N, Ar2-D96N, Ar3-D95N, and Ar4-D98N.21. The system of claim 1, wherein the additional fluorescent protein isVenus, EYFP, or TagRFP.